Cell death regulators

ABSTRACT

A Bcl-2 associated protein (Bax) and uses thereof.

STATEMENT OF RIGHTS

The US Government has a paid-up license in this invention and the rightin limited circumstances to require the patent owner to license otherson reasonable terms as provided for by the terms of Grant No. 49712-05issued by the National Institute of Health.

This is a divisional of application Ser. No. 08/112,208 filed Aug. 26,1993, U.S. Pat. No. 5,691,199.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the identification, purification, and isolationof a novel protein which interacts with Bcl-2 protein to formheteromultimers (heterodimers) in vivo and more particularly to thepurification, isolation and use of Bcl-2 associated protein, hereincalled Bax.

2. Description of the Related Art

Cell death is an important aspect during the embryonic or post-nataldevelopment of major organ systems. Apoptosis, or programmed celldemise, also plays a critical role in maintaining homeostasis in manyadult tissues. Within vertebrates, Bcl-2 is the best understood gene ina cell death pathway and functions as a cell death repressor.

Bcl-2 is unique among protooncogenes by being localized to themitochondrial membrane as defined by Hockenbery, D. M., Nunez, G.,Milliman, C., Schreiber, R. D. and Korsmeyer, S. J. "Bcl-2 is an innermitochondrial membrane protein that blocks programmed cell death."Nature 378, 334-336, 1990. Bcl-2 has been shown to have the oncogenicfunction of blocking programmed cell death whereas a deregulated Bcl-2extends the survival of certain hematopoietic cell lines followinggrowth factor deprivation. When pro-B-cell or promyelocyte cell linesare deprived of interleukin 3 they normally succumb to a programmeddemise entitled apoptosis. This pattern of morphologic cell death ischaracterized by a dramatic plasma membrane blebbing, cell volumecontraction, nuclear pyknosis, and internucleosomal DNA degradationfollowing the activation of an endonuclease. Over expression ofmitochondrial Bcl-2 appears to function as an antidote to this processand has the unique function of blocking programmed cell deathindependent of promoting proliferation.

The Bcl-2 protooncogene was discovered at the chromosomal breakpoint ofthe t(14;18) (q32;q21) which is the cytogenetic hallmark of humanfollicular lymphoma. Approximately 85% of follicular and 20% of diffuseB-cell lymphomas possess this translocation. Follicular lymphoma isoften present as a low-grade malignancy composed of small restingIgM/IgD B cells. Over time, conversion to a more aggressive high-gradelymphoma with a diffuse large-cell architecture frequently occurs.

Studies of Bcl-2 emphasizes the existence of multiple pathways in thegeneration of neoplasia. The increased cell number in neoplastic tissuecan be viewed as a violation of normal homeostasis. The maintenance ofhomeostasis in normal tissue, in many respects, reflects a simplebalanced equation of input (cellular proliferation and renewal) versusoutput (cell death). This is most easily envisioned for encapsulatedorgans, such as the prostate, but is also true of the recirculatinghematopoietic lineages. The maintenance of remarkably invariant cellnumbers reflects tightly regulated death pathways as well as controlledproliferation. See for example S. J. Korsmeyer "Bcl-2 Initiates a NewCategory of Oncogenes: Regulators of Cell Death", Blood Vol. 80 No. 4pp. 879-886, Aug. 15, 1992.

Programmed cell death represents a cell autonomous suicide pathway thathelps restrict cell numbers. The well-defined loss of specific cells iscrucial during embryonic development as part of organogenesis. In maturetissues, genetically programmed demise regulates the volume of cells. Amorphologically distinct and temporally regulated cell death entitledapoptosis has been identified by Wyllie A H: "Apoptosis; Cell death intissue regulation". J. Pathol 153:313, 1987. Cells dying by apoptosisdisplay marked plasma membrane blebbing, volume contraction, nuclearcondensation, and the activation of an endonuclease that cleaves DNAinto nucleosomal length fragments.

Bcl-2 has been localized to chromosome segment 18q21.3 in a telomere tocentromere orientation. The Bcl-2 gene possesses 3 exons, the first ofwhich is untranslated. Two potential promoter regions exist. P1 is GCrich with multiple SP1 sites and is used predominantly. Bcl-2 is anenormous gene in which a 225-kb intron II divides the protein encodingexons II and III. See Silvermann G A et al. "Meiotic recombinationbetween yeast artificial chromosomes yields a single clone containingthe entire Bcl-2 proto-oncogene" Proc Natl Acad Sci 87;9913, 1990. Amolecular consequence of the translation is the movement of the Bcl-2gene to the der(14) chromosome placing Bcl-2 in the same transcriptionalorientation as the Ig heavy chain locus giving rise to chimeric RNAs.However, translocation does not interrupt the protein encoding region sothat normal and translocated alleles produce the same sized, 25-Kdprotein.

Hematopoietic progenitors, including pro-B cells, possess high levels ofBcl-2. See Hockenbery D, Zuter M, Hickey W, Nahm M, Korsmeyer S J:"Bcl-2 protein is topographically restricted in tissues characterized byapoptotic cell death". Proc Natl Acad Sci USA 88:6961, 1991. Some matureB cells and, especially, B-cell lines have low levels of Bcl-2 RNA. Incontrast, t(14;18)-bearing B cells have inappropriate elevated levels ofthe Bcl-2-Ig fusion RNA. Graninger W B, Seto M. Boutain B, Goldman, P,Korsmeyer S J: Expression of Bcl-2 and Bcl-2-Ig fusion transcripts innormal and neoplastic cells. J. Clin Invest 80:1512, 1987. Thisincreased steady-state RNA reflects both increased transcription as wellas a processing advantage for the Bcl-2-Ig fusion allele.

Bcl-2 has been introduced into a variety of interleukin (IL)-dependentcell lines to determine if it is involved in a growth factor pathway.See S J Korsmeyer above. Such lines were examined to determine if Bcl-2would spare the need for a specific ligand/receptor interaction.However, no long-term growth factor-independent cell lines emerged afteroverexpression of Bcl-2 in IL-2, IL-3, IL-4, or IL-6 requiring lines.However, Bcl-2 conferred a death-sparing effect to certain hematopoieticcell lines after growth factor withdrawal in the IL-3-dependent earlyhematopoietic cell lines FDCP1, FL5.12, and 32D. This effect was notrestricted to the IL-3/IL-3 receptor signal transduction pathway in thatgranulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4deprived cells displayed a similar response. Yet, Bcl-2 enhanced cellsurvival was not universal, as neither IL-2-dependent T-cell lines noran IL-6-dependent myeloma line showed a consistent effect upon factorwithdrawal.

Bcl-2 has not been. shown to directly promote cell cycle progression,nor does it necessarily alter the dose response to limitingconcentrations of IL-3. See Nunez G, London L., Hockenbery D, AlexanderM, McKearn J, Korsmeyer S J: "Deregulated Bcl-2 gene expressionselectively prolongs survival of growth factor-deprived hemopoietic celllines". J. Immunol 144;3602, 1990. Instead, Bcl-2 blocked the plasmamembrane blebbing, volume contraction, nuclear condensation, andendonucleolytic cleavage of DNA known as apoptosis. Factor deprivedcells return to Go, but do not die. However, they can be rescued after30 days of deprivation by the addition of IL-3, indicating they are notterminally differentiated or permanently arrested.

While identifying the Bcl-2 cell death pathway is significant, a way ofregulating the Bcl-2 pathway has not been discovered. The ability todown-regulate the effect of Bcl-2 would be advantageous in cancertherapy, in controlling hyperplasias such as benign prostatichypertrophy (BPH) and eliminating self reactive clones in autoimmunityby favoring death effector molecules. Up-regulating the effect of Bcl-2and favoring death repressor molecules would be beneficial in thetreatment and diagnosis of immunodeficiency diseases, including AIDs,and in neurodegenerative and ischemic cell death.

SUMMARY OF THE INVENTION

The present invention relates to the unexpected discovery that Bcl-2interacts with other proteins and in particular with an associated 21 kDprotein called Bax. Bax shares extensive amino acid homology with Bcl-2focused within highly conserved domains I and II. It has beenunexpectedly discovered that Bax homodimerizes and forms heterodimerswith Bcl-2 in vivo. It has also been discovered that overexpressed Baxaccelerates apoptotic death induced by cytokine deprivation in an IL-3dependent cell line and that overexpressed Bax also counters the deathrepressor activity of Bcl-2. This discovery provides a model in whichthe ratio of Bcl-2/Bax determines cell survival or death following anapoptotic stimulus.

Accordingly, one embodiment of the invention involves the formation of apurified and isolated Bcl-2 associated protein (Bax) and fragmentsthereof having the amino acid sequence of Domain I or II of FIG. 7.

Another embodiment involves the formation of Bcl-2 and Bax mutantswherein the native protein or fragment has at least one amino aciddeleted or replaced by another amino acid and the mutants exhibitsaltered biological activity from the native protein or fragment.

Another embodiment involves an associated protein, which comprises Baxprotein coupled with Bcl-2 associated protein or fragments thereof.

A further embodiment involves α DNA isolate consisting essentially of agenomic DNA sequence encoding human Bax and more particularly acomposition consisting of cDNA molecules which encode the Bax protein.

In one aspect of the invention, Bax polypeptides and compositionsthereof are provided. Bax polypeptides comprise polypeptide sequenceswhich are substantially identical to a sequence shown in FIGS. 3, 5 or6. In one embodiment, the Bax polypeptide comprises domain I and/ordomain II of the Bax polypeptide sequence, and preferably comprises theamino acid sequence(s) -W-G-R- and/or -Q-D-Q-, and may be a cyclicpolypeptide in some embodiments.

A further aspect involves a composition of αRNA, βRNA and/or γRNA whichencode a 21 kD, 24 kD or 4 kD Bax protein as well as cell linesproducing such RNA species.

Another aspect of the invention involves Bax pharmaceuticalcompositions, which contain pharmaceutically effective amounts of a Baxpolypeptide and a suitable pharmaceutical carrier.

Polynucleotide sequences encoding Bax are also provided. Thecharacteristics of the cloned sequences are given, including thenucleotide and predicted amino acid sequence in FIGS. 3, 5 and 6.Polynucleotides comprising sequences encoding these amino acid sequencescan serve as templates for the recombinant expression of quantities ofBax polypeptides, such as human Bax and murine Bax.

The invention also provides host cells expressing Bax polypeptidesencoded by a polynucleotide other than a naturally-occurring Bax gene ofthe host cell.

In one aspect of the invention, a polynucleotide encoding a Baxpolypeptide is delivered to a cell, such as an explanted lymphocyte,hematopoietic stem cell, bone marrow cell, and the like.

An additional aspect of the invention involves a method for controllingcell death repressor activity of Bcl-2, which comprises administering aBax protein or fragment thereof to a cell containing Bcl-2 activity toenable the formation of a heterodimer containing Bax/Bcl-2, andinhibiting the Bcl-2 cell death repressor activity.

In one aspect of the invention, a method for modulating apoptosis of acell, typically a lymphocyte, is provided. The method comprisesadministering to a cell an agent which alters intermolecular bindingbetween Bcl-2 and Bax proteins, typically by inhibiting formation ofheteromultimers (e.g., heterodimers) between Bcl-2 and Bax and/orhomomultimers of Bcl-2 or Bax.

In one aspect of the invention, the method(s) of modulating apoptosis ofa cell by administering an agent which alters intermolecular bindingbetween Bcl-2 and Bax proteins are used to treat a pathologicalcondition in a patient.

As an additional embodiment, the invention involves a method forassaying for the predisposition for an apoptotic cell death, whichcomprises: collecting a specimen to be tested; contacting the specimenwith a material reactive with Bcl-2 or Bax protein; and detecting ordetermining the presence or absence of Bax and Bcl-2 protein and theirratio in the specimen.

The invention provides screening assays for identifying agents whichmodulate (e.g., inhibit) binding of a Bax polypeptide to a Bcl-2polypeptide and/or which modulate (e.g., inhibit) binding of a Baxpolypeptide to a Bax polypeptide.

The invention also involves the use of the protein Bax or Bcl-2 ormutant or fragment thereof for performing immunochemical methods for thedetection and determination of the protein or its associated proteinBcl-2, in order to monitor cell survival versus death or to detect ormonitor the course of diseases.

The invention also provides Bax polynucleotide probes for diagnosis ofpathological conditions (e.g., neoplasia, AIDS, hyperplasia, congenitalgenetic diseases) by detection of Bax mRNA or rearrangements deletionsor amplification of the Bax gene in cells explanted from a patient, ordetection of a pathognomonic Bax allele (e.g., by RFLP orallele-specific PCR analysis).

In one aspect of the invention, transgenic nonhuman animals, such asmice, bearing a transgene encoding a Bax polypeptide and/or a Bcl-2polypeptide are provided. Such transgenes may be homologously recombinedinto the host chromosome or may be non-homologously integrated.

Further included is a method for the treatment of a neurodegenerativedisease, an immunodeficiency, or an ischemia, which comprises;increasing the effective amount of Bcl-2 or decreasing Bax oradministering a mutant or fragment thereof to a patient to regulate theratio of Bcl-2 to Bax to promote the survival of cells by generating anexcess of Bcl-2; and, a method for the treatment of hyperplasias,hypertrophies, cancers and autoimmunity disorders, which comprises:decreasing the effective amount of Bcl-2 or increasing Bax oradministering a mutant thereof to a patient to regulate the ratio ofBcl-2 to Bax so as to favor Bax and promote cell death.

In one aspect of the invention, an antisense polynucleotide isadministered to inhibit transcription and/or translation of Bax in acell.

The invention provides antibodies, both monoclonal antibodies andpolyclonal antisera, which specifically bind to a Bax polypeptide withan affinity of about at least 1×10⁷ M⁻¹, typically at least 1×10⁸ M⁻¹ ormore.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the analysis of (³⁵ S) Methionine-labeledimmunoprecipitates using RL-7 cells.

FIG. 2 shows the analysis of (³⁵ S) Methionine-labeledimmunoprecipitates using FL5.12 clones.

FIG. 3 demonstrates the cDNA and protein sequence of human SEQ ID NOS:1and 2! and murine SEQ ID NO:3! Bax.

FIG. 4 demonstrates alternative α, β, γ transcripts and proteins of theBax gene.

FIG. 5 demonstrates the partial nucleotide sequence (SEQ ID NO:4) andamino acid sequence (SEQ ID NO:5) of human Bax βRNA starting at the exon5-intron 5 border of the Bax gene.

FIG. 6 demonstrates the nucleotide sequence (SEQ ID NO:6) and amino acidsequence (SEQ ID NO:7) of human Bax γRNA.

FIG. 7 shows the alignment of the murine and human Bax and Bcl-2proteins. SEQ ID NOS:8 through 11!

FIGS. 8A-8C demonstrate that overexpressed Bax accelerates cell death.

FIGS. 9A-9C demonstrate that the ratio of Bcl-2 and Bax affects theviability of IL-3 deprived FL5.12 cells.

FIG. 10 demonstrates that Bax homodimerizes and forms heterodimers withBcl-2.

FIG. 11 shows western blot analysis of heterodimers.

FIG. 12 shows immunoprecipitations of supernatants.

FIG. 13 demonstrates the interrelationship between Bcl-2 and Bax and theregulation of programmed cell death.

FIG. 14 shows in FIG. 14A the Domain I and II regions in a family ofBcl-2 closely related genes and in FIGS. 14B, 14C the mutations inDomains I and II, respectively of the Bcl-2 mutants generated and testedfor binding and death repressor activity.

FIGS. 15A-15D show an analysis of the level of mutant Bcl-2 protein intwo cell lines (FL5.12 and 2B4).

FIGS. 16A-16C show an IL-3 deprivation time course of stable celltransfects and DNA Fragmentation assay (FL5.12).

FIGS. 17A-17B show two viability studies of cell lines (2B4).

FIGS. 18A-18B show immunoprecipitations of radiolabeled transfectants.

FIGS. 19A-19D demonstrate a parallel assessment of stable transfectantsof domain II Bcl-2 mutants.

FIGS. 20A-20D demonstrate the cell death response in cells with domainII Bcl-2 mutants and 2 immunoprecipitations of radiolabeled transfectedcells.

FIGS. 21A-21C show cells that have two expression constructs andindicates the effect of Bcl-2 domain I mutations on homodimerization andheterodimerization.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage (Immunology--A Synthesis, 2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991), which is incorporated herein by reference).Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention. Similarly,unless specified otherwise, the lefthand end of single-strandedpolynucleotide sequences is the 5' end; the lefthand direction ofdouble-stranded polynucleotide sequences is referred to as the 5'direction. The direction of 5' to 3' addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5' to the5' end of the RNA transcript are referred to as "upstream sequences";sequence regions on the DNA strand having the same sequence as the RNAand which are 3' to the 3' end of the RNA transcript are referred to as"downstream sequences".

The term "naturally-occurring" as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

As used herein, the term "Bax" refers to the mammalian Bax gene andmammalian Bax proteins, including the α, β, and γ isoforms, unlessotherwise identified; human and murine Bax proteins and genes arepreferred exemplifications of mammalian Bax, and in its narrowest usageBax refers to human Bax polynucleotide and polypeptide sequences.

The term "corresponds to" is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term "complementary to" is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence "TATAC" corresponds to a reference sequence "TATAC"and is complementary to a reference sequence "GTATA".

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: "reference sequence", "comparisonwindow", "sequence identity", "percentage of sequence identity", and"substantial identity". A "reference sequence" is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence of FIG. 3 or FIGS. 5 and 6, or may comprise acomplete cDNA or gene sequence. Generally, a reference sequence is atleast 20 nucleotides in length, frequently at least 25 nucleotides inlength, and often at least 50 nucleotides in length. Since twopolynucleotides may each (1) comprise a sequence (i.e., a portion of thecomplete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a "comparison window" toidentify and compare local regions of sequence similarity.

A "comparison window", as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

The term "sequence identity" means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term "percentage of sequence identity" is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms "substantial identity" as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 85 percentsequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence, for example, as a segment of thefull-length human Bax polynucleotide sequence shown in FIG. 3 or FIGS. 5and 6.

As applied to polypeptides, the term "substantial identity" means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are; valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term "Bax native protein" and "full-length Bax protein" as usedherein refers to a full-length Bax α, β, or γ isoform of 192 aminoacids, 218 amino acids, or 41 amino acids as shown herein (see, FIG. 4).A preferred Bax native protein is the polypeptide corresponding to thededuced amino acid sequence shown in FIG. 3 or corresponding to thededuced amino acid sequence of a cognate full-length cDNA of anotherspecies. Also for example, a native Bax protein present innaturally-occurring lymphocytes which express the Bax gene areconsidered full-length Bax proteins.

The term "fragment" as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence deduced from a full-length cDNA sequence (e.g., the cDNAsequence shown in FIG. 3). Fragments typically are at least 14 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer.

The term "analog", "mutein" or "mutant" as used herein refers topolypeptides which are comprised of a segment of at least 10 amino acidsthat has substantial identity to a portion of the naturally occurringprotein. For example, a Bax analog comprises a segment of at least 10amino acids that has substantial identity to a Bax protein, such as thehuman α, β, or γ isoforms; preferably the deduced amino acid sequenceshown in FIG. 3 or deduced amino acid sequences shown in FIGS. 5 and 6,and which has at least one of the following properties: binding to Bcl-2or native Bax protein under suitable binding conditions. Typically,analog polypeptides comprise a conservative amino acid substitution (oraddition or deletion) with respect to the naturally-occurring sequence.Analogs typically are at least 20 amino acids long, preferably at least50 amino acids long or longer, most usually being as long as full-lengthnaturally-occurring protein (e.g., 192, 218, or 41 amino acid residuesfor human Bax α, β, and γ, respectively) Some analogs may lackbiological activity (e.g., Bcl-2 binding) but may still be employed forvarious uses, such as for raising antibodies to Bax epitopes, as animmunological reagent to detect and/or purify α-Bax antibodies byaffinity chromatography, or as a competitive or noncompetitive agonist,antagonist, or partial agonist of native Bax protein function.

The term "Bax polypeptide" is used herein as a generic term to refer tonative protein, fragments, or analogs of Bax. Hence, native Bax,fragments of Bax, and analogs of Bax are species of the Bax polypeptidegenus. Preferred Bax polypeptides include: a murine full-length Baxprotein comprising the murine polypeptide sequence shown in FIG. 3, afull-length human Bax protein comprising the polypeptide sequence shownin FIG. 3, polypeptides consisting essentially of the sequence of humanBax domain I or domain II, and the naturally-occurring human Bax α, β,and γ isoforms.

The term "Bcl-2 polypeptide" is used herein as a generic term to referto native protein, fragments, or analogs of Bcl-2, preferably human ormurine Bcl-2, usually human Bcl-2.

The term "cognate" as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example butnot limitation, in the human genome, the human CD4 gene is the cognategene to the mouse CD4 gene, since the sequences and structures of thesetwo genes indicate that they are highly homologous and both genes encodea protein which functions in signaling T cell activation through MHCclass II-restricted antigen recognition. Thus, the cognate human gene tothe murine Bax gene is the human gene which encodes an expressed isprotein which has the greatest degree of sequence identity to the murineBax protein and which exhibits an expression pattern similar to that ofthe murine Bax (e.g., expressed in lymphocytes). Preferred cognate Baxgenes are: rat Bax, rabbit Bax, canine Bax, nonhuman primate Bax,porcine Bax, bovine Bax, and hamster Bax.

The term "agent" is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues. Agents are evaluated forpotential activity as antineoplastics or apoptosis modulators byinclusion in screening assays described hereinbelow.

The term "antineoplastic agent" is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a lymphocytic leukemia, lymphomaor pre-leukemic condition.

As used herein, the terms "label" or "labeled" refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes (e.g., ³ H, ¹⁴ C, ³⁵ S ¹²⁵ I, ¹³¹ I), fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), biotinyl groups, predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, "substantially pure" means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

As used herein "normal blood" or "normal human blood" refers to bloodfrom a healthy human individual who does not have an active neoplasticdisease or other disorder of lymphocytic proliferation, or an identifiedpredisposition for developing a neoplastic disease. Similarly, "normalcells", "normal cellular sample", "normal tissue", and "normal lymphnode" refers to the respective sample obtained from a healthy humanindividual who does not have an active neoplastic disease or otherlymphoproliferative disorder.

As used herein the terms "pathognomonic concentration", "pathognomonicamount", and "pathognomonic staining pattern" refer to a concentration,amount, or localization pattern, respectively, of a Bax protein or mRNAin a sample, that indicates the presence of a pathological (e.g.,neoplastic) condition or a predisposition to developing a neoplasticdisease, such as lymphocytic leukemia. A pathognomonic amount is anamount of a Bax protein or Bax mRNA in a cell or cellular sample thatfalls outside the range of normal clinical values that is established byprospective and/or retrospective statistical clinical studies.Generally, an individual having a neoplastic disease (e.g., lymphocyticleukemia) will exhibit an amount of Bax protein or mRNA in a cell ortissue sample that is outside the range of concentrations thatcharacterize normal, undiseased individuals; typically the pathognomonicconcentration is at least about one standard deviation outside the meannormal value, more usually it is at least about two standard deviationsor more above the mean normal value. However, essentially all clinicaldiagnostic tests produce some percentage of false positives and falsenegatives. The sensitivity and selectivity of the diagnostic assay mustbe sufficient to satisfy the diagnostic objective and any relevantregulatory requirements. In general, the diagnostic methods of theinvention are used to identify individuals as disease candidates,providing an additional parameter in a differential diagnosis of diseasemade by a competent health professional.

Oligonucleotides can be synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Methods for PCR amplification are described in the art (PCR Technology:Principles and Applications for DNA Amplification ed. H A Erlich,Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guide to Methodsand Applications, eds. Innis, Gelfland, Snisky, and White, AcademicPress, San Diego, Calif. (1990); Mattila et al. (1991) Nucleic AcidsRes. 19: 4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods andApplications 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; and U.S. Pat. No. 4,683,202, which are incorporated herein byreference).

It is known that the development as well as the maintenance of manyadult tissues is achieved by several dynamically regulated processesthat include cell proliferation, differentiation and programmed celldeath. In the latter process, cells are eliminated by a highlycharacteristic suicide program entitled apoptosis.

Bcl-2 was first isolated at the chromosomal breakpoint of t(14;18)bearing follicular B cell lymphomas. Transgenic mice bearing a Bcl-2-Igmini-gene that recapitulates this translocation display a polyclonalfollicular hyperplasia with a four-fold increase in resting B cells andas such B cells accumulate because of extended cell survival rather thanincreased proliferation.

A survey of adult tissues indicates that Bcl-2 has played several rolesin numerous cell lineages. Glandular epithelium that undergoeshyperplasia or involution in response to hormonal stimuli or growthfactors express Bcl-2. In complex epithelium, such as the skin and gut,Bcl-2 is restricted to stem cells and proliferation zones. Within theadult nervous system Bcl-2 is more prominent in the peripheral nervoussystem rather than the central nervous system. Thus Bcl-2 may be neededto save the progenitor and long-lived cells in a variety of celllineages.

Despite the progress in defining Bcl-2's physiologic roles, thebiochemical basis of its action has remained ambiguous up until thepresent invention. Dual fluorescence staining of cells, examined with alaser scanning confocal microscope, indicates that Bcl-2 protein withina B cell line was coincident with the distribution of mitochondria.Subcellular fractionation studies revealed that the majority of Bcl-2was localized as an integral mitochondrial membrane protein whichsuggests that Bcl-2 might alter some mitochondrial function associatedwith energy production. However, Bcl-2 was able to prevent cell death ina fibroblast line that lacks mitochondrial DNA.

Bcl-2 appears to function in several subcellular locations, yet lacksany known motifs that would confer a biochemical role. It has beenunexpectedly discovered that Bcl-2 associates, in vivo with a 21 kDprotein partner, herein called Bax. Bax shows extensive amino acidhomology with Bcl-2 and forms homodimers with itself and heterodimerswith Bcl-2 in vivo. Bax is encoded by 6 exons and demonstrates a complexpattern of alternative RNA splicing that predicts a 21 Kd membrane (α)and two forms (β and γ) of cytosolic protein. When Bax predominates,programmed cell death is accelerated and the death repressor activity ofBcl-2 is countered.

According to the present invention a co-immunoprecipitation procedurewas used to identify Bax, the novel protein associated with Bcl-2. Itwas completely unexpected to find that Bax shared extensive homologywith Bcl-2, especially within two highly conserved domains. Thesedomains are also the most highly conserved regions of human, mouse andchicken Bcl-2. These domains are also conserved in an open reading frameBHRF-1 within Epstein-Barr virus and Mcl-1, a gene recently isolatedfrom a myeloid leukemia cell line following induction with phorbol ester(Kozopas et al., 1993). Thus, a clear family of Bcl-2-like genes isappearing and are likely to be sequential numbers of a single deathpathway or regulators of parallel death pathways.

As discussed above, Bax homodimerizes or heterodimerizes with Bcl-2 invivo. While the precise multiplicity of these interactions is not fullyknown, conserved domains I and II are areas of dimerization motifs. Baxαpossesses a COOH-terminal hydrophobic segment predicted to be membranespanning and has a similar secondary structure to Bcl-2. Thus, the twoproteins are likely to be inserted into the same membrane with anidentical orientation. However, coinsertion is not required forassociation in that the ΔC-22 cytosolic form of Bcl-2 stillcoprecipitates Baxα, provided it has been solubilized from membranes.Since Bcl-2 ΔC-22 partially protects cells from death, this furtherstrengthens the importance of the Bcl-2/Bax interaction. Moreover,results using FL5.12 cells indicate that Bax homodimerization is favoredover heterodimerization with Bcl-2. It has been found that the majorityof introduced HA-Baxα dimerizes with endogenous Baxα rather than withthe modest amounts of endogenous Bcl-2 in FL5.12 cells. Overexpressionof Bcl-2 in these cells thus competes for Bax homodimerization and formsheterodimers with HA-Baxα and endogenous Baxα.

The complexity of the RNA splicing therein proves to be an importantdifferential regulator of Bax activity and localization. The predictedmembrane α and cytosolic β forms of Bax are parallel to the α integralmembrane form of Bcl-2 and predicted β cytosolic form. The exon/intronjuncture responsible for the α and β RNAs are evolutionarily conservedand thus the existence of 24 kD Baxβ protein is believed to be proven.Consistent with this, RL-7 cells display a 24 kD protein associated withBcl-2 and possess the 1.5 kb βRNA, while FL5.12 cells lack both. Acytosolic Baxβ could provide an additional level of regulation byhomodimerizing or heterodimerizing with the integral membrane forms ofBax and Bcl-2. The γ form of Bax RNA might result in a truncated γprotein or could represent a splicing strategy to avoid making the fulllength product.

It has been discovered that the ratio of Bcl-2/Bax determines a cellssusceptibility to death following an apoptotic stimulus. In the presenceof IL-3 overexpressed Bax does not noticeably alter normal cell divisionor viability. Bax is present and associated with Bcl-2 prior to growthfactor deprivation. Moreover, the ratio of Bcl-2/Bax within FL5.12 cellsis not substantially altered 12 hrs after deprivation of IL-3. Bax RNAis expressed in normal tissues and in a variety of cell lines prior to adeath induction signal. Thus, the synthesis of Bax does not appear to bea denovo response that follows a death stimulus, and Bax in itselfaccelerates apoptotic cell death only following a death signal, such asIL-3 deprivation. Excess Bax also counters the death repressor activityof Bcl-2. When Bcl-2 is in excess cells are protected. However, when Baxis in excess and Bax homodimers dominate, cells are susceptible toapoptosis.

It has also been discovered that a single amino acid substitution inBcl-2 eliminates Bcl-2/Bax heterodimers but not Bcl-2 homodimers andabolishes death repressor activity. As discussed herein, the Bcl-2proto-oncogene inhibits apoptosis induced by a variety of signals withinmultiple cell types. Protein mutations with as minor as a single aminoacid substitution within one conserved region, domain I, of the Bcl-2molecule also eliminates its death repressor activity. Mutated Bcl-2 nolonger blocks cell death induced by factor deprivation, glucocorticoidtreatment or gamma irradiation. Bcl-2 mutations that no longer function,no longer heterodimerize with Bax, and when overexpressed accelerateprogrammed cell death. Mutations within domain II of Bcl-2 partiallydisrupt its death repressor activity and correspondingly partiallyinhibit its heterodimerization with Bax. However, mutant Bcl-2 as wellas wild-type Bcl-2 still effectively forms homodimers. These resultsdocument the importance of the conserved sequences especially domain Iand their role in heterodimerization for Bcl-2 and Bax. Such domains arealso likely to be instrumental in dictating homo and heterodimerizationformation in other Bcl-2 family members including Bcl-x, MCL-1, LMW5-HLand BHRF1. The current data support a model in which Bcl-2 functions byneutralizing a death accelerating protein Bax throughheterodimerization. Therapeutic modalities which disrupt Bcl-2/Baxheterodimers prove profoundly effective in promoting cell death.

Several mechanistic possibilities are believed to account for theregulatory role of this protein-protein interaction. Bax might functionas a death effector molecule that is neutralized by Bcl-2. In thisscenario, Bcl-2 might simply be an inert handcuff that disrupts theformation of Bax homodimers. Alternatively, Bcl-2 could possess abiochemical function that is diametrically opposed to Bax. In contrastBcl-2 might function as a death repressor molecule that is neutralizedby competition with an inert Bax molecule. Either way, the capacity ofBax and Bcl-2 to compete for one another via heterodimers indicates areciprocal relationship in which Bcl-2 monomers or homodimers favorsurvival, and Bax homodimers favor death.

Mammalian cells are often dependent upon an extracellular milieuincluding growth and survival factors or cell--cell contact molecules.The dependence of the early hematopoietic cell line, FL5.12, upon IL-3for its survival as well as proliferation is a typical example. However,a number of biologic systems indicate that cells within the same lineagehave an inherent sensitivity or resistance to a given death stimulus.For example, CD4⁺ 8⁺ cortical thymocytes are sensitive to glucocorticoidinduced apoptosis while the more mature medullary thymocytes areresistant. This differential sensitivity correlates with the presence ofBcl-2 protein. The Bcl-2/Bax interaction represents one such endogenousregulator of susceptibility to apoptosis. These discoveries suggest amodel in which the response of a cell to a death signal is determined bya preset mechanism, such as the ratio of Bcl-2/Bax.

Because of this interaction it is possible to use this invention for thedetection and determination of Bax or Bcl-2, for example in a fractionfrom a tissue or organ separation operation, or immunochemical techniquein view of the proteins antigenic properties. Specific antibodies canalso be formed on immunization of animals with this protein. It is knownthat monoclonal antibodies already exist to Bcl-2.

An antiserum which can be utilized for this purpose can be obtained byconventional procedures. One exemplary procedure involves theimmunization of a mammal, such as rabbits, which induces the formationof polyclonal antibodies against Bax. Monoclonal antibodies are alsobeing generated from already immunized hamsters. This antibody can beused to detect the presence and level of the Bax protein.

It is also possible to use the proteins for the immunological detectionof Bax, Bcl-2 and associations thereof with standard assays as well asassays using markers, which are radioimmunoassays or enzymeimmunoassays.

The detection and determination of Bax and/or Bcl-2 has significantdiagnostic importance. For example, the detection of proteins favoringdeath effector molecules would be advantageous in cancer therapy andcontrolling hypertrophies and eliminating self reactive clones inautoimmunity. The detection or determination of proteins favoring deathrepressor molecules will be beneficial in immunodeficiency disease,including HIV-I, II and III, and in neurodegenerative and ischemic celldeath. Thus these proteins and their antibodies can be employed as amarker to monitor, check or detect the course of disease.

More particularly, the protein Bax may be used for performingimmunochemical methods for the detection and determination of theprotein or its associated protein Bcl-2, in order to monitor cell growthor to detect or monitor the course of diseases. It can also be used as amethod for the treatment of a neurodegenerative disease, orimmunodeficiency, or an ischemia induced injury such as myocardialinfarction and neurologic stroke, which comprises; administering aneffective amount of a compound to a patient to regulate the ratio ofBcl-2 to Bax to promote the survival of cells by generating an excess ofBcl-2.

A method for the treatment of hyperplasias, hypertrophies, cancers andautoimmunity disorders, which comprises: administering an effectiveamount of a compound to a patient to regulate the ratio of Bcl-2 to Baxso as to favor the Bax protein and promote cell death.

Specific preparations of the Bax proteins and compounds can also beprepared for administration in pharmaceutical preparations. These may beaccomplished in a variety of ways well known to those skilled in the artof pharmacy.

It will be understood that the precise chemical structure of Bax andBcl-2 will depend upon a number of factors. For example, since ionizableamino and carboxyl groups are present in the molecule, a particularprotein may be obtained as an acidic or basic salt, or in neutral form.All forms of Bax which retain their therapeutic activity for purposes ofthe instant invention are intended to be within the scope of thedefinition of Bax.

The term "recombinant" used herein refers to Bax and Bcl-2 produced byrecombinant DNA techniques wherein the gene coding for protein is clonedby known recombinant DNA technology. For example, the human gene for Baxmay be inserted into a suitable DNA vector, such as a bacterial plasmid,and the plasmid used to transform a suitable host. The gene is thenexpressed in the host to produce the recombinant protein. Thetransformed host may be prokaryotic or eukaryotic, including mammalian,yeast, Aspergillus and insect cells. One preferred embodiment employsbacterial cells as the host.

Therapeutically useful derivatives of Bax may be prepared by augmentingthe primary amino acid sequence of the protein with at least oneadditional molecule selected from the group consisting of glucosemoieties, lipids, phosphate groups, acetyl groups, hydroxyl groups,saccharides, methyl groups, propyl groups, amino acids, and polymericmolecules. Augmentation may be accomplished through post-translationalprocessing systems of the producing host, or it may be carried out invitro. Both techniques are well-known in the art.

Referring to the Sequence Description of Bax, it should be noted thatthe peptide includes several potential glycosylation sites.Glycosylation is a process of forming a protein derivative, wherein aportion of the protein's amino acid sequence is augmented by a sugarmoiety. It will therefore be understood that therapeutically usefulderivatives of Bax may be prepared by addition of one or more sugarresidues to the protein, or alternatively by removal of some or all ofthe sugar residues from the sites of glycosylation on the Bax molecule.

Other therapeutically useful derivatives of Bax may be formed bymodifying at least one amino acid residue of Bax or Bcl-2 by oxidation,reduction, or other derivatization processes known in the art.

Mutants of Bax and Bcl-2 which modify the activity of the protein may beused as the active treating substance of the instant invention. Muteinsare prepared by modification of the primary structure of the proteinitself, by deletion, addition, or alteration of the amino acidsincorporated into the sequence during translation. For example, at leastone glycine residue of Bax or Bcl-2 in Domain I may be replaced with anamino acid such as alanine or glutamic acid. Also, it may be desirableto eliminate or replace a group of amino aids, such as the FRDG or WGRsequence in Bcl-2 domain I to remove bioactivity of the protein.

Bax and Bcl-2 are believed to exist in nature as a dimer of twoidentical, non-covalently linked protein subunits. Accordingly, sinceeach subunit is believed to have the amino acid sequence shown in SEQ IDNO:1 and 2, a subunit of Bax or Bcl-2 could be used as thetherapeutically active or diagnostic substance according to the instantinvention. The invention also encompasses use of subunits of proteinthat are covalently or non-covalently linked, either naturally or byartificial techniques known in the art.

In addition, it is contemplated that fragments of Bax and Bcl-2 would beuseful in the invention, provided that such fragments retained theirtherapeutic activity.

The fragment defined by amino acid residues in domain I and domain II ofFIG. 7 are believed to be therapeutically active for purposes of theinvention. The fragment defined by these residues is believed to betherapeutically active because: it is a linear sequence not involvingdisulfide bridges and because it appears to be key to repressing celldeath.

The above-described forms of Bax and Bcl-2 are used in an effectivetherapeutic amount, which will vary depending on the level of Bax andBcl-2 already present in the patient, the site and method ofadministration, the form of protein utilized, and other factorsunderstood to those having ordinary skill in the art.

Cloning of Bax Polynucleotides

Genomic or cDNA clones encoding Bax may be isolated from clone libraries(e.g., available from Clontech, Palo Alto, Calif.) using hybridizationprobes designed on the basis of the nucleotide sequences shown in FIG. 3and FIGS. 5 and 6 and using conventional hybridization screening methods(e.g., Benton W D and Davis R W (1977) Science 196: 180; Goodspeed etal. (1989) Gene 76: 1). Where a cDNA clone is desired, clone librariescontaining cDNA derived from lymphocyte mRNA or other Bax-expressingcell mRNA are preferred. Alternatively, synthetic polynucleotidesequences corresponding to all or part of the sequences shown in FIG. 3and FIGS. 5 and 6 may be constructed by chemical synthesis ofoligonucleotides. Additionally, polymerase chain reaction (PCR) usingprimers based on the sequence data disclosed in FIG. 3 and FIGS. 5 and 6may be used to amplify DNA fragments from genomic DNA, mRNA pools, orfrom cDNA clone libraries. U.S. Pat. Nos. 4,683,195 and 4,683,202describe the PCR method. Additionally, PCR methods employing one primerthat is based on the sequence data disclosed in FIG. 3 and asecondprimer that is not based on that sequence data may be used. Forexample, a second primer that is homologous to or complementary to apolyadenylation segment may be used.

It is apparent to one of skill in the art that nucleotide substitutions,deletions, and additions may be incorporated into the polynucleotides ofthe invention. Nucleotide sequence variation may result from sequencepolymorphisms of various Bax alleles, minor sequencing errors, and thelike. However, such nucleotide substitutions, deletions, and additionsshould not substantially disrupt the ability of the polynucleotide tohybridize to one of the polynucleotide sequences shown in FIG. 3 orFIGS. 5 and 6 under hybridization conditions that are sufficientlystringent to result in specific hybridization.

Specific hybridization is defined herein as the formation of hybridsbetween a probe polynucleotide (e.g., a polynucleotide of the inventionwhich may include substitutions, deletion, and/or additions) and aspecific target polynucleotide (e.g., a polynucleotide having thesequence in FIG. 3 or FIGS. 5 and 6), wherein the probe preferentiallyhybridizes to the specific target such that, for example, a single bandcorresponding to one or more of the isoforms of Bax (α, β, or γ)alternatively spliced mRNA species can be identified on a Northern blotof RNA prepared from a suitable cell source (e.g., a T or B cellexpressing Bax) Polynucleotides of the invention and recombinantlyproduced Bax, and fragments or analogs thereof, may be prepared on thebasis of the sequence data provided in FIG. 3 and FIGS. 5 and 6according to methods known in the art and described in Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989), Cold SpringHarbor, N.Y. and Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif., which are incorporated herein by reference.

Bax polynucleotides may be short oligonucleotides (e.g., 25-100 baseslong), such as for use as hybridization probes and PCR (or LCR) primers.Bax polynucleotide sequences may also comprise part of a largerpolynucleotide (e.g., a cloning vector comprising a Bax clone) and maybe fused, by polynucleotide linkage, in frame with anotherpolynucleotide sequence encoding a different protein (e.g., glutathioneS-transferase or β-galactosidase) for encoding expression of a fusionprotein. Typically, Bax polynucleotides comprise at least 25 consecutivenucleotides which are substantially identical to a naturally-occurringBax sequence (e.g., FIG. 3), more usually Bax polynucleotides compriseat least 50 to 100 consecutive nucleotides which are substantiallyidentical to a naturally-occurring Bax sequence. However, it will berecognized by those of skill that the minimum length of a Baxpolynucleotide required for specific hybridization to a Bax targetsequence will depend on several factors: G/C content, positioning ofmismatched bases (if any), degree of uniqueness of the sequence ascompared to the population of target polynucleotides, and chemicalnature of the polynucleotide (e.g., methylphosphonate backbone,phosphorothiolate, etc.), among others.

If desired, PCR amplimers for amplifying substantially full-length cDNAcopies may be selected at the discretion of the practitioner. Similarly,amplimers to amplify single Bax exons or portions of the Bax gene(murine or human) may be selected.

Each of these sequences may be used as hybridization probes or PCRamplimers to detect the presence of Bax mRNA, for example to diagnose alymphoproliferative disease characterized by the presence of an elevatedor reduced Bax mRNA level in lymphocytes, or to perform tissue typing(i.e., identify tissues characterized by the expression of Bax mRNA),and the like. The sequences may also be used for detecting genomic Baxgene sequences in a DNA sample, such as for forensic DNA analysis (e.g.,by RFLP analysis, PCR product length(s) distribution, etc.) or fordiagnosis of diseases characterized by amplification and/orrearrangements of the Bax gene.

Production of Bax Polypeptides

The nucleotide and amino acid sequences shown in FIG. 3 and FIG. 5 and 6enable those of skill in the art to produce polypeptides correspondingto all or part of the full-length human and murine Bax polypeptidesequences. Such polypeptides may be produced in prokaryotic oreukaryotic host cells by expression of polynucleotides encoding Bax, orfragments and analogs thereof. Alternatively, such polypeptides may besynthesized by chemical methods or produced by in vitro translationsystems using a polynucleotide template to direct translation. Methodsfor expression of heterologous proteins in recombinant hosts, chemicalsynthesis of polypeptides, and in vitro translation are well known inthe art and are described further in Maniatis et al., Molecular Cloning:A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y. and Bergerand Kimmel, Methods in Enzymology, Volume 152, Guide to MolecularCloning Techniques (1987), Academic Press, Inc., San Diego, Calif.

Fragments or analogs of Bax may be prepared by those of skill in theart. Preferred amino- and carboxy-termini of fragments or analogs of Baxoccur near boundaries of functional domains. For example, but not forlimitation, such functional domains include domains conferring theproperty of binding to a Bcl-2 polypeptide, and (2) domains conferringthe property of binding to a Bax polypeptide.

One method by which structural and functional domains may be identifiedis by comparison of the nucleotide and/or amino acid sequence data shownin FIG. 3 or FIGS. 5 and 6 to public or proprietary sequence databases.Preferably, computerized comparison methods are used to identifysequence motifs or predicted protein conformation domains that occur inother proteins of known structure and/or function, such as domain I anddomain II. For example, the NAD-binding domains of dehydrogenases,particularly lactate dehydrogenase and malate dehydrogenase, are similarin conformation and have amino acid sequences that are detectablyhomologous (Proteins, Structures and Molecular Principles, (1984)Creighton (ed.), W. H. Freeman and Company, New York, which isincorporated herein by reference). Further, a method to identify proteinsequences that fold into a known three-dimensional structure are known(Bowie et al. (1991) Science 253: 164). Thus, the foregoing examplesdemonstrate that those of skill in the art can recognize sequence motifsand structural conformations that may be used to define structural andfunctional domains in the Bax sequences of the invention.

Additionally, computerized comparison of sequences shown in FIG. 3 orFIGS. 5 and 6 to existing sequence databases can identify sequencemotifs and structural conformations found in other proteins or codingsequences that indicate similar domains of the Bax protein. For examplebut not for limitation, the programs GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package (Genetics Computer Group, 575Science Dr., Madison, Wis.) can be used to identify sequences indatabases, such as GenBank/EMBL, that have regions of homology with aBax sequences. Such homologous regions are candidate structural orfunctional domains. Alternatively, other algorithms are provided foridentifying such domains from sequence data. Further, neural networkmethods, whether implemented in hardware or software, may be used to:(1) identify related protein sequences and nucleotide sequences, and (2)define structural or functional domains in Bax polypeptides (Brunak etal. (1991) J. Mol. Biol. 220: 49, which is incorporated herein byreference).

Fragments or analogs comprising substantially one or more functionaldomain may be fused to heterologous polypeptide sequences, wherein theresultant fusion protein exhibits the functional property(ies) conferredby the Bax fragment. Alternatively, Bax polypeptides wherein one or morefunctional domain have been deleted will exhibit a loss of the propertynormally conferred by the missing fragment.

By way of example and not limitation, the domain(s) conferring theproperty of binding to Bcl-2 may be fused to β-galactosidase to producea fusion protein that can bind an immobilized Bcl-2 polypeptide in abinding reaction and which can enzymatically convert a chromogenicsubstrate to a chromophore.

Although one class of preferred embodiments are fragments having amino-and/or carboxy-termini corresponding to amino acid positions nearfunctional domains borders, alternative Bax fragments may be prepared.The choice of the amino- and carboxy-termini of such fragments restswith the discretion of the practitioner and will be made based onexperimental considerations such as ease of construction, stability toproteolysis, thermal stability, immunological reactivity, amino- orcarboxyl-terminal residue modification, or other considerations.

In addition to fragments, analogs of Bax can be made. Such analogs mayinclude one or more deletions or additions of amino acid sequence,either at the amino- or carboxy-termini, or internally, or both; analogsmay further include sequence transpositions. Analogs may also compriseamino acid substitutions, preferably conservative substitutions.Additionally, analogs may include heterologous sequences generallylinked at the amino- or carboxy-terminus, wherein the heterologoussequence(s) confer a functional property to the resultant analog whichis not indigenous to the native Bax protein. However, Bax analogs mustcomprise a segment of 25 amino acids that has substantial similarity toa portion of the amino acid sequences shown in FIG. 3 or FIGS. 5 and 6or other mammalian Bax proteins, respectively, and which has at leastone of the requisite functional properties (i.e., forms heterodimerswith Bcl-2 and/or forms homodimers with Bax. Preferred amino acidsubstitutions are those which: (1) reduce susceptibility to proteolysis,(2) reduce susceptibility to oxidation, (3) alter post-translationalmodification of the analog, possibly including phosphorylation, and (4)confer or modify other physicochemical or functional properties of suchanalogs. Bax analogs include various muteins of a Bax sequence otherthan the naturally-occurring peptide sequence. For example, single ormultiple amino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally-occurring Bax sequence(preferably in the portion of the polypeptide outside domains I and II).

Conservative amino acid substitution is a substitution of an amino acidby a replacement amino acid which has similar characteristics (e.g.,those with acidic properties: Asp and Glu). A conservative (orsynonymous) amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles, (1984) Creighton (ed.), W.H.Freeman and Company, New York; Introduction to Protein Structure,(1991), C. Branden and J. Tooze, Garland Publishing, New York, N.Y.; andThornton et al. (1991) Nature 354: 105; which are incorporated herein byreference).

Similarly, full-length Bcl-2 polypeptides and fragments or analogsthereof can be made by those of skill in the art from the availableBcl-2 gene, cDNA, and protein sequences (e.g., GenBank).

Native Bax proteins, fragments thereof, or analogs thereof can be usedas reagents in binding assays to detect binding to Bcl-2 and or bindingto Bax for identifying agents that interfere with Bax function, saidagents are thereby identified as candidate drugs which may be used, forexample, to block apoptosis, to induce apoptosis (e.g., to treatlymphocytic leukemias), and the like. Typically, in vitro binding assaysthat measure binding of Bax to Bcl-2 employ native Bax (α or β isoform)that contains domain I and domain II. The Bcl-2 (or Bax) polypeptide istypically linked to a solid substrate by any of various means known tothose of skill in the art; such linkage may be noncovalent (e.g.,binding to a highly charged surface such as Nylon 66) or may be bycovalent bonding (e.g., typically by chemical linkage). Bax polypeptidesare typically labeled by incorporation of a radiolabeled amino acid orfluorescent label. The labeled Bax polypeptide is contacted with theimmobilized Bcl-2 (or Bax) polypeptide under aqueous conditions thatpermit specific binding in control binding reactions with a bindingaffinity of about 1×10⁵ M⁻¹ or greater (e.g., 10-250 mM NaCl or is KCland 5-100 mM Tris HCl pH 5-9, usually pH 6-8), generally including Zn⁺²and/or Mn⁺² and/or Mg⁺² in the nanomolar to micromolar range (1 nM to999 μM). Specificity of binding is typically established by addingunlabeled competitor at various concentrations selected at thediscretion of the practitioner. Examples of unlabeled proteincompetitors include, but are not limited to, the following: unlabeledBax polypeptide, bovine serum albumin, and cellular protein extracts.Binding reactions wherein one or more agents are added are performed inparallel with a control binding reaction that does not include an agent.Agents which inhibit the specific binding of Bax polypeptides to Bcl-2polypeptides and/or Bax polypeptides, as compared to a control reaction,are identified as candidate Bax modulating drugs.

Peptidomimetics

In addition to Bax polypeptides consisting only of naturally-occurringamino acids, Bax peptidomimetics are also provided. Peptide analogs arecommonly used in the pharmaceutical industry as non-peptide drugs withproperties analogous to those of the template peptide. These types ofnonpeptide compound are termed "peptide mimetics" or "peptidomimetics"(Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985)TINS p.392; and Evans et al. (1987) J. Med. Chem 30: 1229, which areincorporated herein by reference) and are usually developed with the aidof computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biological or pharmacological activity),such as human Bax, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: --CH₂ NH--,--CH₂ S--, --CH₂ --CH₂ --, --CH═CH-- (cis and trans), --COCH₂ --,--CH(OH)CH₂ --, and --CH₂ SO--, by methods known in the art and furtherdescribed in the following references: Spatola, A. F. in "Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein,eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data(March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (generalreview); Morley, J. S., Trends Pharm Sci (1980) pp. 463-468 (generalreview); Hudson, D. et al., Int J Pept Prot Res (1979) 14:177-185 (--CH₂NH--, CH₂ CH₂ --); Spatola, A. F. et al., Life Sci (1986) 38:1243-1249(--CH₂ --S); Hann, M. M., J Chem Soc Perkin Trans I (1982) 307-314(--CH--CH--, cis and trans); Almquist, R. G. et al., J Med Chem (1980)23:1392-1398 (--COCH₂ --); Jennings-White, C. et al., Tetrahedron Lett(1982) 23:2533 (--COCH₂ --); Szelke, M. et al., European Appln. EP 45665(1982) CA: 97:39405 (1982) (--CH(OH)CH₂ --); Holladay, M. W. et al.,Tetrahedron Lett (1983) 24:4401-4404 (--C(OH)CH₂ --); and Hruby, V. J.,Life Sci (1982) 31:189-199 (--CH₂ --S--); each of which is incorporatedherein by reference. A particularly preferred non-peptide linkage is--CH₂ NH--. Such peptide mimetics may have significant advantages overpolypeptide embodiments, including, for example: more economicalproduction, greater chemical stability, enhanced pharmacologicalproperties (half-life, absorption, potency, efficacy, etc.), alteredspecificity (e.g., a broad-spectrum of biological activities), reducedantigenicity, and others. Labeling of peptidomimetics usually involvescovalent attachment of one or more labels, directly or through a spacer(e.g., an amide group), to non-interfering position(s) on thepeptidomimetic that are predicted by quantitative structure-activitydata and/or molecular modeling. Such non-interfering positions generallyare positions that do not form direct contacts with themacromolecules(s) to which the peptidomimetic binds to produce thetherapeutic effect. Derivitization (e.g., labelling) of peptidomimeticsshould not substantially interfere with the desired biological orpharmacological activity of the peptidomimetic.

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387,incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide. Cyclic peptides comprising the sequence -WGR-and/or -QDN- and/or -FRDG- frequently are preferred.

The amino acid sequences of Bax polypeptides identified herein willenable those of skill in the art to produce polypeptides correspondingto Bax peptide sequences and sequence variants thereof. Suchpolypeptides may be produced in prokaryotic or eukaryotic host cells byexpression of polynucleotides encoding a Bax peptide sequence,frequently as part of a larger polypeptide. Alternatively, such peptidesmay be synthesized by chemical methods. Methods for expression ofheterologous proteins in recombinant hosts, chemical synthesis ofpolypeptides, and in vitro translation are well known in the art and aredescribed further in Maniatis et al., Molecular Cloning: A LaboratoryManual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel,Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques(1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969)J. Am. Chem. Soc. 91: 501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem.11: 255; Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986)Science 232: 342; Kent, S. B. H. (1988) Ann. Rev. Biochem. 57: 957; andOfford, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which areincorporated herein by reference).

Peptides of the sequence N-W-G-R or W-G-R can be produced, typically bydirect chemical synthesis, and used as agents to competitively inhibitBax/Bcl-2 heterodimers formation. The N-W- G-R and W-G-R peptides arefrequently produced as modified peptides, with nonpeptide moietiesattached by covalent linkage to the N-terminus and/or C-terminus. Incertain preferred embodiments, either the carboxy-terminus or theamino-terminus, or both, are chemically modified. The most commonmodifications of the terminal amino and carboxyl groups are acetylationand amidation, respectively. Amino-terminal modifications such asacylation (e.g., acetylation) or alkylation (e.g., methylation) andcarboxy-terminal-modifications such as amidation, as well as otherterminal modifications, including cyclization, may be incorporated intovarious embodiments of the invention. Certain amino-terminal and/orcarboxy-terminal modifications and/or peptide extensions to the coresequence can provide advantageous physical, chemical, biochemical, andpharmacological properties, such as: enhanced stability, increasedpotency and/or efficacy, resistance to serum proteases, desirablepharmacokinetic properties, and others. Such N-W-G-R and W-G-R peptidesmay be used therapeutically to treat disease by altering the process ofapoptosis in a cell population of a patient.

Production and Applications of α-Bax Antibodies

Native Bax proteins, fragments thereof, or analogs thereof, may be usedto immunize an animal for the production of specific antibodies. Theseantibodies may comprise a polyclonal antiserum or may comprise amonoclonal antibody produced by hybridoma cells. For general methods toprepare antibodies, see Antibodies: A Laboratory Manual, (1988) E.Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., which is incorporated herein by reference.

For example but not for limitation, a recombinantly produced fragment ofhuman Bax can be injected into a mouse along with an adjuvant followingimmunization protocols known to those of skill in the art so as togenerate an immune response. Typically, approximately at least 1-50 μgof a Bax fragment or analog is used for the initial immunization,depending upon the length of the polypeptide. Alternatively or incombination with a recombinantly produced Bax polypeptide, a chemicallysynthesized peptide having a Bax sequence may be used as an immunogen toraise antibodies which bind a Bax protein, such as the native human Baxpolypeptide having the sequence shown essentially in FIG. 3 or thenative human Bax polypeptide having the sequence shown essentially inFIGS. 5 and 6. Immunoglobulins which bind the recombinant fragment witha binding affinity of at least 1×10⁷ M⁻¹ can be harvested from theimmunized animal as an antiserum, and may be further purified byimmunoaffinity chromatography or other means. Additionally, spleen cellsare harvested from the immunized animal (typically rat or mouse) andfused to myeloma cells to produce a bank of antibody-secreting hybridomacells. The bank of hybridomas can be screened for clones that secreteimmunoglobulins which bind the recombinantly produced Bax polypeptide(or chemically synthesized Bax polypeptide) with an affinity of at least1×10⁶ M⁻¹. Animals other than mice and rats may be used to raiseantibodies; for example, goats, rabbits, sheep, and chickens may also beemployed to raise antibodies reactive with a Bax protein. Transgenicmice having the capacity to produce substantially human antibodies alsomay be immunized and used for a source of α-Bax antiserum and/or formaking monoclonal-secreting hybridomas.

Bacteriophage antibody display libraries may also be screened forbinding to a Bax polypeptide, such as a full-length human Bax protein, aBax fragment, or a fusion protein comprising a Bax polypeptide sequencecomprising a Bax epitope (generally at least 3-5 contiguous aminoacids). Generally such Bax peptides and the fusion protein portionsconsisting of Bax sequences for screening antibody libraries compriseabout at least 3 to 5 contiguous amino acids of Bax, frequently at least7 contiguous amino acids of Bax, usually comprise at least 10 contiguousamino acids of Bax, and most usually comprise a Bax sequence of at least14 contiguous amino acids as shown in FIG. 3 or FIGS. 5 and 6.Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which may be screened asbacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:2432). Various embodiments of bacteriophage antibody display librariesand lambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991)Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147:3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol.Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992)Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007;Lowman et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science258: 1313, incorporated herein by reference). Typically, a bacteriophageantibody display library is screened with a Bax polypeptide that isimmobilized (e.g., by covalent linkage to a chromatography resin toenrich for reactive phage by affinity chromatography) and/or labeled(e.g., to screen plaque or colony lifts).

Bax polypeptides which are useful as immunogens, for diagnosticdetection of α-Bax antibodies in a sample, for diagnostic detection andquantitation of Bax protein in a sample (e.g., by standardizedcompetitive ELISA), or for screening a bacteriophage antibody displaylibrary, are suitably obtained in substantially pure form, that is,typically about 50 percent (w/w) or more purity, substantially free ofinterfering proteins and contaminants. Preferably, these polypeptidesare isolated or synthesized in a purity of at least 80 percent (w/w)and, more preferably, in at least about 95 percent (w/w) purity, beingsubstantially free of other proteins of humans, mice, or othercontaminants.

For some applications of these antibodies, such as identifyingimmunocrossreactive proteins, the desired antiserum or monoclonalantibody(ies) is/are not monospecific. In these instances, it may bepreferable to use a synthetic or recombinant fragment of Bax as anantigen rather than using the entire native protein. More specifically,where the object is to identify immunocrossreactive polypeptides thatcomprise a particular structural moiety, such as a Bcl-2 binding domain,it is preferable to use as an antigen a fragment corresponding to partor all of a commensurate structural domain in the Bax protein.Production of recombinant or synthetic fragments having such definedamino- and carboxy-termini is provided by the Bax sequences shown inFIG. 3 and FIGS. 5 and 6.

If an antiserum is raised to a Bax fusion polypeptide, such as a fusionprotein comprising a Bax immunogenic epitope fused to β-galactosidase orglutathione S-transferase, the antiserum is preferably preadsorbed withthe non-Bax fusion partner (e.g, β-galactosidase or glutathioneS-transferase) to deplete the antiserum of antibodies that react (i.e.,specifically bind to) the non-Bax portion of the fusion protein thatserves as the immunogen. Monoclonal or polyclonal antibodies which bindto the human and/or murine Bax protein can be used to detect thepresence of human or murine Bax polypeptides in a sample, such as aWestern blot of denatured protein (e.g., a nitrocellulose blot of anSDS-PAGE) obtained from a lymphocyte sample of a patient. Preferablyquantitative detection is performed, such as by denistometric scanningand signal integration of a Western blot. The monoclonal or polyclonalantibodies will bind to the denatured Bax epitopes and may be identifiedvisually or by other optical means with a labeled second antibody orlabeled Staphylococcus aureus protein A by methods known in the art.

One use of such antibodies is to screen cDNA expression libraries,preferably containing cDNA derived from human or murine mRNA fromvarious tissues, for identifying clones containing cDNA inserts whichencode structurally-related, immunocrossreactive proteins, that arecandidate novel Bcl-2 binding factors or Bax-related proteins. Suchscreening of cDNA expression libraries is well known in the art, and isfurther described in Young et al., Proc. Natl. Acad. Sci. U.S.A.80:1194-1198 (1983), which is incorporated herein by reference! as wellas other published sources. Another use of such antibodies is toidentify and/or purify immunocrossreactive proteins that arestructurally or evolutionarily related to the native Bax protein or tothe corresponding Bax fragment (e.g., functional domain; Bcl-2-bindingdomain) used to generate the antibody. The anti-Bax antibodies of theinvention can be used to measure levels of Bax protein in a cell or cellpopulation, for example in a cell explant (e.g., lymphocyte sample)obtained from a patient. When used in conjunction with antibodies thatspecifically bind to Bcl-2, the anti-Bax antibodies of the presentinvention can be used to measure the ratio of Bax protein to Bcl-2protein (i.e., Bax:Bcl-2 ratio) in a cell or cell population. Theanti-Bax and anti-Bcl-2 antibodies can be used to measure thecorresponding protein levels (Bax or Bcl-2, respectively) by variousmethods, including but not limited to: (1) standardized ELISA on cellextracts, (2) immunoprecipitation of cell extracts followed bypolyacrylamide gel electrophoresis of the immunoprecipitated productsand quantitative detection of the band(s) corresponding to Bax and/orBcl-2, and (3) in situdetection by immmunohistochemical straining withthe anti-Bax and/or anti-Bcl-2 antibodies and detection with a labeledsecond antibody. The measurement of the Bax:Bcl-2 ratio in a cell orcell population is informative regarding the apoptosis status of thecell or cell population.

Various other uses of such antibodies are to diagnose and/or stageleukemias or other neoplasms, and for therapeutic application (e.g., ascationized antibodies or by targeted liposomal delivery) to treatneoplasia, autoimmune disease, AIDS, and the like.

Bax Polynucleotides

Disclosure of the full coding sequences for murine and human Bax shownin FIG. 3 and FIGS. 5 and 6 makes possible the construction of isolatedpolynucleotides that can direct the expression of Bax, fragmentsthereof, or analogs thereof. Further, the sequences in FIG. 3 and FIGS.5 and 6 make possible the construction of nucleic acid hybridizationprobes and PCR primers that can be used to detect RNA and DNA sequencesencoding Bax.

Polynucleotides encoding full-length Bax or fragments or analogsthereof, may include sequences that facilitate transcription (expressionsequences) and translation of the coding sequences, such that theencoded polypeptide product is produced. Construction of suchpolynucleotides is well known in the art and is described further inManiatis et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989),Cold Spring Harbor, N.Y. For example, but not for limitation, suchpolynucleotides can include a promoter, a transcription termination site(polyadenylation site in eukaryotic expression hosts), a ribosomebinding site, and, optionally, an enhancer for use in eukaryoticexpression hosts, and, optionally, sequences necessary for replicationof a vector. A typical eukaryotic expression cassette will include apolynucleotide sequence encoding a Bax polypeptide linked downstream(i.e., in translational reading frame orientation; polynucleotidelinkage) of a promoter such as the HSV tk promoter or the pgk(phosphoglycerate kinase) promoter, optionally linked to an enhancer anda downstream polyadenylation site (e.g., an SV40 large T Ag poly Aaddition site).

Preferably, these amino acid sequences occur in the given order (in theamino terminal to carboxyterminal orientation) and may comprise otherintervening and/or terminal sequences; generally such polypeptides areless than 1000 amino acids in length, more usually less than about 500amino acids in lengths, and frequently approximately 41 to 218 aminoacids in length (e.g., 192 amino acids or 218 amino acids; α or β humanisoforms). The degeneracy of the genetic code gives a finite set ofpolynucleotide sequences encoding these amino acid sequences; this setof degenerate sequences may be readily generated by hand or by computerusing commercially available software (Wisconsin Genetics SoftwarePackage Relaes 7.0). Isolated Bax polynucleotides typically are lessthan approximately 10,000 nucleotides in length.

Additionally, where expression of a polypeptide is not desired,polynucleotides of this invention need not encode a functional protein.Polynucleotides of this invention may serve as hybridization probesand/or PCR primers (amplimers) and/or LCR oligomers for detecting BaxRNA or DNA sequences.

Alternatively, polynucleotides of this invention may serve ashybridization probes or primers for detecting RNA or DNA sequences ofrelated genes, such genes may encode structurally or evolutionarilyrelated proteins. For such hybridization and PCR applications, thepolynucleotides of the invention need not encode a functionalpolypeptide. Thus, polynucleotides of the invention may containsubstantial deletions, additions, nucleotide substitutions and/ortranspositions, so long as specific hybridization or specificamplification to a Bax sequence is retained.

Specific hybridization is defined hereinbefore, and can be roughlysummarized as the formation of hybrids between a polynucleotide of theinvention (which may include substitutions, deletions, and/or additions)and a specific target polynucleotide such as murine or human Bax mRNA sothat a single band corresponding to each isoform present is identifiedon a Northern blot of RNA prepared from Bax-expressing cells (i.e.,hybridization and washing conditions can be established that permitdetection of discrete Bax mRNA band(s)). Thus, those of ordinary skillin the art can prepare polynucleotides of the invention, which mayinclude substantial additions, deletions, substitutions, ortranspositions of nucleotide sequence as compared to sequences shown inFIG. 3 or FIGS. 5 and 6, and determine whether specific hybridization isa property of the polynucleotide by performing a Northern blot using RNAprepared from a lymphocyte cell line which expresses Bax mRNA and/or byhybridization to a Bax DNA clone (cDNA or genomic clone).

Specific amplification is defined as the ability of a set of PCRamplimers, when used together in a PCR reaction with a Baxpolynucleotide, to produce substantially a single major amplificationproduct which corresponds to a Bax gene sequence or mRNA sequence.Generally, human genomic DNA or mRNA from Bax expressing human cells(e.g., Jurkat cell line) is used as the template DNA sample for the PCRreaction. PCR amplimers that exhibit specific amplification are suitablefor quantitative determination of Bax mRNA by quantitative PCRamplification. Bax allele-specific amplification products, althoughhaving sequence and/or length polymorphisms, are considered toconstitute a single amplification product for purposes of thisdefinition.

Generally, hybridization probes comprise approximately at least 10 andpreferably 25 consecutive nucleotides of a sequence shown in FIG. 3 orFIGS. 5 and 6 (for human and murine Bax detection), preferably thehybridization probes contain at least 50 consecutive nucleotides of asequence shown in FIG. 3 or FIGS. 5 and 6, and more preferably compriseat least 100 consecutive nucleotides of a sequence shown in FIG. 3 orFIGS. 5 and 6. PCR amplimers typically comprise approximately 25 to 50consecutive nucleotides of a sequence shown in FIG. 3 or FIGS. 5 and 6,and usually consist essentially of approximately 25 to 50 consecutivenucleotides of a sequence shown in FIG. 3 or FIGS. 5 and 6 withadditional nucleotides, if present, generally being at the 5' end so asnot to interfere with polymerase-mediated chain extension. PCR amplimerdesign and hybridization probe selection are well within the scope ofdiscretion of practitioners of ordinary skill in the art.

Methods of Identifying Novel and Apoptosis-Modulating Agents

A basis of the present invention is the experimental finding that anovel protein, Bax, is present in many cell types which undergoapoptosis and Bax binds specifically to Bcl-2, a protein known tomodulate (inhibit) apoptosis in cells. For example, agents which blockBax function and/or block Bcl-2 function may be developed as potentialhuman therapeutic drugs.

Therapeutic agents which inhibit cell death by modulatingBcl-2-dependent inhibition of Bax function (i.e., formation of Bax/Baxhomodimers and/or induction of apoptosis), for example by augmentingformation of Bcl-2/Bax heterodimers and thereby reducing formation ofBax/Bax homodimers, can be used as pharmaceuticals. Such pharmaceuticalswill be used to treat a variety of human a veterinary diseases, such as:reperfusion injury, myocardial infarction, stroke, traumatic braininjury, neurodegenerative diseases, aging, ischemia, toxemia, infection,AIDS, hepatitis, and the like.

Therapeutic agents which augment (induce) cell death by modulating thelevels of Bax/Bcl-2 heterodimers and Bax/Bax homodimers can be used aspharmaceuticals. Such pharmaceuticals can be used to treat a variety ofdiseases including but not limited to: hyperplasia, neoplasia,autoimmune diseases, transplant rejection, lymphoproliferative diseases,and the like.

Candidate antineoplastic agents are then tested further forantineoplastic activity in assays which are routinely used to predictsuitability for use as human antineoplastic drugs. Examples of theseassays include, but are not limited to: (1) ability of the candidateagent to inhibit the ability of anchorage-independent transformed cellsto grow in soft agar, (2) ability to reduce tumorigenicity oftransformed cells transplanted into nu/nu mice, (3) ability to reversemorphological transformation of transformed cells, (4) ability to reducegrowth of transplanted tumors in nu/nu mice, (5) ability to inhibitformation of tumors or preneoplastic cells in animal models ofspontaneous or chemically-induced carcinogenesis, and (6) ability toinduce a more differentiated phenotype in transformed cells to which theagent is applied.

Bax/Bcl-2 Intermolecular Binding

A basis of the present invention is the surprising finding that the Baxprotein forms a complex with the Bcl-2 protein under physiologicalconditions. This finding indicates that the Bax protein serves as amodulator of Bcl-2 function, and vice versa. Such functional modulationcan serve to couple a signal transduction pathway (via Bax) to anapoptosis regulatory protein (i.e., Bcl-2).

Assays for detecting the ability of agents to inhibit or augment thebinding of Bax to Bcl-2 provide for facile high-throughput screening ofagent banks (e.g., compound libraries, peptide libraries, and the like)to identify Bax or Bcl-2 antagonists or agonists. Such Bax or Bcl-2antagonists and agonists may modulate Bax and/or Bcl-2 activity andthereby modulate apoptosis.

Administration of an efficacious dose of an agent capable ofspecifically inhibiting Bax/Bcl-2 complex formation or Bcl-2/Bcl-2complex formation to a patient can be used as a therapeutic orprophylactic method for treating pathological conditions (e.g., cancer,inflammation, lymphoproliferative diseases, autoimmune disease,neurodegenerative diseases, and the like) which are effectively treatedby modulating Bax and/or Bcl-2 activity and apoptosis.

Binding assays generally take one of two forms: immobilized Baxpolypeptide(s) can be used to bind labeled Bcl-2 polypeptide(s), orconversely, immobilized Bcl-2 polypeptide(s) can be used to bind labeledBax polypeptides. Alternatively, a binding assay can be performed todetect binding of a Bax polypeptide to form a homodimer with a Baxpolypeptide; typically, a labeled Bax polypeptide is contacted with animmobilized Bax polypeptide under aqueous binding conditions and theextent of binding is determined by measuring the amount of immobilizedlabeled Bax. In each case, the labeled polypeptide is contacted with theimmobilized polypeptide under aqueous conditions that permit specificbinding of the polypeptides(s) to form a Bax/Bcl-2 complex in theabsence of added agent. Particular aqueous conditions may be selected bythe practitioner according to conventional methods. For generalguidance, the following buffered aqueous conditions may be used: 10-250mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of divalentcation(s) and/or metal chelators and/or nonionic detergents and/ormembrane fractions. It is appreciated by those in the art thatadditions, deletions, modifications (such as pH) and substitutions (suchas KCl substituting for NaCl or buffer substitution) may be made tothese basic conditions. Modifications can be made to the basic bindingreaction conditions so long as specific binding of Bax polypeptide(s) toBcl-2 polypeptides occurs in the control reaction(s). In someembodiments, where the assay detects formation of Bax/Bax homodimers,modifications can be made to the basic binding reaction conditions solong as specific binding of a Bax polypeptide to a Bax polypeptidesoccurs in the control reaction(s). Conditions that do not permitspecific binding in control reactions (no agent included) are notsuitable for use in binding assays.

Preferably, at least one polypeptide species is labeled with adetectable marker. Suitable labeling includes, but is not limited to,radiolabeling by incorporation of a radiolabeled amino acid (e.g., ¹⁴C-labeled leucine, ³ H-labeled glycine, ³⁵ S-labeled methionine),radiolabeling by post-translational radioiodination with ¹²⁵ I or ¹³¹ I(e.g., Bolton-Hunter reaction and chloramine T), labeling bypost-translational phosphorylation with ³² P (e.g., phosphorylase andinorganic radiolabeled phosphate) fluorescent labeling by incorporationof a fluorescent label (e.g., fluorescein or rhodamine), or labeling byother conventional methods known in the art. In embodiments where one ofthe polypeptide species is immobilized by linkage to a substrate, theother polypeptide is generally labeled with a detectable marker.

Additionally, in some embodiments a Bax or Bcl-2 polypeptide may be usedin combination with an accessory protein (e.g., a protein which forms acomplex with the polypeptide in vivo), it is preferred that differentlabels are used for each polypeptide species, so that binding ofindividual and/or heterodimeric and/or multimeric complexes can bedistinguished. For example but not limitation, a Bax polypeptide may belabeled with fluorescein and an accessory polypeptide may be labeledwith a fluorescent marker that fluorescesces with either a differentexcitation wavelength or emission wavelength, or both. Alternatively,double-label scintillation counting may be used, wherein a Baxpolypeptide is labeled with one isotope (e.g., ³ H) and a secondpolypeptide species is labeled with a different isotope (e.g., ¹⁴ C)that can be distinguished by scintillation counting using discriminationtechniques.

Labeled polypeptide(s) are contacted with immobilized polypeptide(s)under aqueous conditions as described herein. The time and temperatureof incubation of a binding reaction may be varied, so long as theselected conditions permit specific binding to occur in a controlreaction where no agent is present. Preferable embodiments employ areaction temperature of about at least 15 degrees Centigrade, morepreferably 35 to 42 degrees Centigrade, and a time of incubation ofapproximately at least 15 seconds, although longer incubation periodsare preferable so that, in some embodiments, a binding equilibrium isattained. Binding kinetics and the thermodynamic stability of boundBax/Bcl-2 complexes determine the latitude available for varying thetime, temperature, salt, pH, and other reaction conditions. However, forany particular embodiment, desired binding reaction conditions can becalibrated readily by the practitioner using conventional methods in theart, which may include binding analysis using Scatchard analysis, Hillanalysis, and other methods (Proteins, Structures and MolecularPrinciples, (1984) Creighton (ed.), W.H. Freeman and Company, New York).

Specific binding of labeled Bax or Bcl-2 polypeptide to immobilizedBcl-2 or Bax polypeptide, respectively, is determined by includingunlabeled competitor protein(s) (e.g., albumin). After a bindingreaction is completed, labeled polypeptide(s) that is/are specificallybound to immobilized polypeptide is detected. For example and not forlimitation, after a suitable incubation period for binding, the aqueousphase containing non-immobilized protein is removed and the substratecontaining the immobilized polypeptide species and any labeled proteinbound to it is washed with a suitable buffer, optionally containingunlabeled blocking agent(s), and the wash buffer(s) removed. Afterwashing, the amount of detectable label remaining specifically bound tothe immobilized polypeptide is determined (e.g., by optical, enzymatic,autoradiographic, or other radiochemical methods).

In some embodiments, addition of unlabeled blocking agents that inhibitnon-specific binding are included. Examples of such blocking agentsinclude, but are not limited to, the following: calf thymus DNA, salmonsperm DNA, yeast RNA, mixed sequence (random or pseudorandom sequence)oligonucleotides of various lengths, bovine serum albumin, nonionicdetergents (NP-40, Tween, Triton X-100, etc.), nonfat dry milk proteins,Denhardt's reagent, polyvinylpyrrolidone, Ficoll, and other blockingagents. Practitioners may, in their discretion, select blocking agentsat suitable concentrations to be included in binding assays; however,reaction conditions are selected so as to permit specific bindingbetween a Bax polypeptide and a Bcl-2 polypeptide in a control bindingreaction. Blocking agents are included to inhibit nonspecific binding oflabeled protein to immobilized protein and/or to inhibit nonspecificbinding of labeled polypeptide to the immobilization substrate.

In embodiments where a polypeptide is immobilized, covalent ornoncovalent linkage to a substrate may be used. Covalent linkagechemistries include, but are not limited to, well-characterized methodsknown in the art (Kadonaga and Tijan (1986) Proc. Natl. Acad. Sci.(U.S.A.) 83: 5889). One example, not for limitation, is covalent linkageto a substrate derivatized with cyanogen bromide (such asCNBr-derivatized Sepharose 4B). It may be desirable to use a spacer toreduce potential steric hindrance from the substrate. Noncovalentbonding of proteins to a substrate include, but are not limited to,bonding of the protein to a charged surface and binding with specificantibodies.

In one class of embodiments, parallel binding reactions are conducted,wherein one set of reactions serves as control and at least one otherset of reactions include various quantities of agents, mixtures ofagents, or biological extracts, that are being tested for the capacityto inhibit binding of a Bax polypeptide to a Bcl-2 polypeptide, and/orto inhibit binding of a Bax polypeptide to form homomultimers(homodimers) with a Bax polypeptide.

Yeast Two-Hybrid Screening Assays

Yeast comprising (1) an expression cassette encoding a GAL4 DNA bindingdomain (or GAL4 activator domain) fused to a binding fragment of Bcl-2capable of binding to a Bax polypeptide, (2) an expression cassetteencoding a GAL4 DNA activator domain (or GAL4 binding domain,respectively) fused to a binding fragment of Bax capable of binding to aBcl-2 polypeptide, and (3) a reporter gene (e.g., β-galactosidase)comprising a cis-linked GAL4 transcriptional response element can beused for agent screening. Such yeast are incubated with a test agent andexpression of the reporter gene (e.g., β-galactosidase) is determined;the capacity of the agent to inhibit expression of the reporter gene ascompared to a control culture identifies the agent as a candidateBcl-2-modulatory agent or Bax modulatory agent.

Yeast two-hybrid systems may be used to screen a mammalian (typicallyhuman) cDNA expression library, wherein cDNA is fused to a GAL4 DNAbinding domain or activator domain, and either a Bax or Bcl-2polypeptide sequence is fused to a GAL4 activator domain or DNA bindingdomain, respectively. Such a yeast two-hybrid system can screen forcDNAs that encode proteins which bind to Bax or Bcl-2 sequences. Forexample, a cDNA library can be produced from mRNA from a human mature Bcell (Namalwa) line (Ambrus et al. (1993) Proc. Natl. Acad. Sci.(U.S.A.)) or other suitable cell type. Such a cDNA library cloned in ayeast two-hybrid expression system (Chien et al. (1991) Proc. Natl.Acad. Sci. (U.S.A.) 88: 9578) can be used to identify cDNAs which encodeproteins that interact with Bax or Bcl-2 and thereby produce expressionof the GAL4-dependent reporter gene. Polypeptides which interact withBax or Bcl-2 can also be identified by immunoprecipitation of Bax orBcl-2 with antibody and identification of co-precipitating species.Further, polypeptides that bind Bax or Bcl-2 can be identified byscreening a peptide library (e.g., a bacteriophage peptide displaylibrary, a spatially defined VLSIPS peptide array, and the like) with aBax or Bcl-2 polypeptide.

Antisense Polynucleotides

Additional embodiments directed to modulation of neoplasia or apoptosisinclude methods that employ specific antisense polynucleotidescomplementary to all or part of the sequences shown in FIG. 3 or FIGS. 5and 6. Such complementary antisense polynucleotides may includenucleotide substitutions, additions, deletions, or transpositions, solong as specific hybridization to the relevant target sequencecorresponding to FIG. 3 or FIGS. 5 and 6 is retained as a functionalproperty of the polynucleotide. Complementary antisense polynucleotidesinclude soluble antisense RNA or DNA oligonucleotides which canhybridize specifically to Bax mRNA species and prevent transcription ofthe mRNA species and/or translation of the encoded polypeptide (Ching etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10006; Broder et al. (1990)Ann. Int. Med. 113: 604; Loreau et al. (1990) FEBS Letters 274: 53;Holcenberg et al., WO91/11535; U.S. Ser. No. 07/530,165; WO91/09865;WO91/04753; WO90/13641; and EP 386563, each of which is incorporatedherein by reference). The antisense polynucleotides therefore inhibitproduction of Bax polypeptides. Antisense polynucleotides that preventtranscription and/or translation of mRNA corresponding to Baxpolypeptides may inhibit apoptosis, senescence, AIDS, and the like,and/or reverse the transformed phenotype of cells. Antisensepolynucleotides of various lengths may be produced, although suchantisense polynucleotides typically comprise a sequence of about atleast 25 consecutive nucleotides which are substantially identical to anaturally-occurring Bax polynucleotide sequence, and typically which areidentical to a sequence shown in FIG. 3 or FIGS. 5 or 6.

Antisense polynucleotides may be produced from a heterologous expressioncassette in a transfectant cell or transgenic cell, such as a transgenicpluripotent hematopoietic stem cell used to reconstitute all or part ofthe hematopoietic stem cell population of an individual. Alternatively,the antisense polynucleotides may comprise soluble oligonucleotides thatare administered to the external milieu, either in the culture medium invitro or in the circulatory system or interstitial fluid in vivo.Soluble antisense polynucleotides present in the external milieu havebeen shown to gain access to the cytoplasm and inhibit translation ofspecific mRNA species. In some embodiments the antisense polynucleotidescomprise methylphosphonate moieties. For general methods relating toantisense polynucleotides, see Antisense RNA and DNA, (1988) D. A.Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Transgenic Animal Embodiments

Genomic clones of Bax, particularly of the murine cognate Bax gene, maybe used to construct homologous targeting constructs for generatingcells and transgenic nonhuman animals having at least one functionallydisrupted Bax allele. Guidance for construction of homologous targetingconstructs may be found in the art, including: Rahemtulla et al. (1991)Nature 353: 180; Jasin et al. (1990) Genes Devel. 4: 157; Koh et al.(1992) Science 256: 1210; Molina et al. (1992) Nature 357: 161; Grusbyet al. (1991) Science 253: 1417; Bradley et al. (1992) Bio/Technology10: 534. Homologous targeting can be used to generate so-called"knockout" mice, which are heterozygous or homozygous for an inactivatedBax allele. Such mice may be sold commercially as research animals forinvestigation of immune system development, neoplasia, apoptosis, andother uses.

Chimeric targeted mice are derived according to Hogan, et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed., IRL Press, Washington, D.C.,(1987). Embryonic stem cells are manipulated according to publishedprocedures (Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed., IRL Press, Washington, D.C. (1987);Zjilstra et al. (1989) Nature 342:435; and Schwartzberg et al. (1989)Science 246: 799.

Additionally, a Bax cDNA or genomic gene copy may be used to constructtransgenes for expressing Bax polypeptides at high levels and/or underthe transcriptional control of transcription control sequences which donot naturally occur adjacent to the Bax gene. For example but notlimitation, a constitutive promoter (e.g., a CMV or pgk promoter) or acell-lineage specific transcriptional regulatory sequence (e.g., an LCKor immunoglobulin gene promoter/enhancer) have been operably linked to aBax-encoding polynucleotide sequence to form a transgene. Suchtransgenes can be introduced into cells (e.g., fertilized eggs, EScells, hematopoietic stem cells) and transgenic cells and transgenicnonhuman animals may be obtained according to conventional methods.Transgenic cells and/or transgenic nonhuman animals may be used toscreen for antineoplastic agents and/or to screen for potentialcarcinogens or agents that modulate apoptosis, as overexpression of Baxor inappropriate expression of Bax may result in a preneoplastic orneoplastic state, may prevent neoplastic development, or may producepremature senescence or depletion or ablation of specific lymphocytecompartments.

Identification and Isolation of Proteins That Bind Bax

Proteins that bind to Bax and/or a Bax/Bcl-2 complex are potentiallyimportant regulatory proteins. Such proteins may be targets for novelantineoplastic agents and other novel drugs. These proteins are referredto herein as accessory proteins. Accessory proteins may be isolated byvarious methods known in the art.

One preferred method of isolating accessory proteins is by contacting aBax polypeptide in a cell extract to an antibody that binds the Baxpolypeptide, and isolating resultant immune complexes. These immunecomplexes may contain accessory proteins bound to the Bax polypeptide.The accessory proteins may be identified and isolated by denaturing theimmune complexes with a denaturing agent and, preferably, a reducingagent. The denatured, and preferably reduced, proteins can beelectrophoresed on a polyacrylamide gel. Putative accessory proteins canbe identified on the polyacrylamide gel by one or more of various wellknown methods (e.g., Coomassie staining, Western blotting, silverstaining, etc.), and isolated by resection of a portion of thepolyacrylamide gel containing the relevant identified polypeptide andelution of the polypeptide from the gel portion.

Yeast two-hybrid systems wherein on GAL4 fusion protein comprises a Baxpolypeptide sequence, typically a full-length of near full-length Baxpolypeptide sequence (e.g., the sequence of FIG. 3), and the other GAL4fusion protein comprises a cDNA library member can be used to identifycDNAs encoding Bax-interacting proteins, according to the general methodof Chien et al. (1991) op.cit. Alternatively, an E. coli/BCCPinteractive screening system (Germino et al. (1993) Proc. Natl. Acad.Sci. (U.S.A.) 90: 1639, incorporated herein by reference) can be used toidentify Bax-interacting protein sequences. Also, an expression library,such as λgtll cDNA expression library (Dunn et al. (1989) J. Biol. Chem.264: 13057), can be screened with a labelled Bax polypeptide to identifycDNAs encoding polypeptides which specifically bind Bax. For theseprocedures, cDNA libraries usually comprise mammalian cDNA populations,typically human, mouse, or rat, and may represent cDNA produced from RNAand one cell type, tissue, or organ and one or more developmental stage.Specific binding for screening cDNA expression libraries is usuallyprovided by including one or more blocking agent (e.g., albumin, nonfatdry milk solids, etc.) prior to and/or concomitant with contacting thelabeled Bax polypeptide (and/or labeled anti-Bax antibody).

A putative accessory protein may be identified as an accessory proteinby demonstration that the protein binds to Bax and/or a Bax/Bcl-2complex. Such binding may be shown in vitro by various means, including,but not limited to, binding assays employing a putative accessoryprotein that has been renatured subsequent to isolation by apolyacrylamide gel electrophoresis method. Alternatively, binding assaysemploying recombinant or chemically synthesized putative accessoryprotein may be used. For example, a putative accessory protein may beisolated and all or part of its amino acid sequence determined bychemical sequencing, such as Edman degradation. The amino acid sequenceinformation may be used to chemically synthesize the putative accessoryprotein. The amino acid sequence may also be used to produce arecombinant putative accessory protein by: (1) isolating a cDNA cloneencoding the putative accessory protein by screening a cDNA library withdegenerate oligonucleotide probes according to the amino acid sequencedata, (2) expressing the cDNA in a host cell, and (3) isolating theputative accessory protein.

Putative accessory proteins that bind Bax and/or Bax/Bcl-2 complex invitro are identified as accessory proteins. Accessory proteins may alsobe identified by crosslinking in vivo with bifunctional crosslinkingreagents (e.g., dimethylsuberimidate, glutaraldehyde, etc.) andsubsequent isolation of crosslinked products that include a Baxpolypeptide. For a general discussion of cross-linking, see Kunkel etal. (1981) Mol. Cell. Biochem. 34:3. Preferably, the bifunctionalcrosslinking reagent will produce crosslinks which may be reversed underspecific conditions after isolation of the crosslinked complex so as tofacilitate isolation of the accessory protein from the Bax polypeptide.Isolation of crosslinked complexes that include a Bax polypeptide ispreferably accomplished by binding an antibody that binds a Baxpolypeptide with an affinity of at least 1×10⁷ M⁻¹ to a population ofcrosslinked complexes and recovering only those complexes that bind tothe antibody with an affinity of at least 1×10⁷ M⁻¹. Polypeptides thatare crosslinked to a Bax polypeptide are identified as accessoryproteins.

Screening assays can be developed for identifying candidateantineoplastic agents as being agents which inhibit binding of Bax to anaccessory protein under suitable binding conditions.

Methods for Forensic Identification

The Bax polynucleotide sequences of the present invention can be usedfor forensic identification of individual humans, such as foridentification of decedents, determination of paternity, criminalidentification, and the like. For example but not limitation, a DNAsample can be obtained from a person or from a cellular sample (e.g.,crime scene evidence such as blood, saliva, semen, and the like) andsubjected to RFLP analysis, allele-specific PCR, or PCR cloning andsequencing of the amplification product to determine the structure ofthe Bax gene region. On the basis of the Bax gene structure, theindividual from which the sample originated will be identified withrespect to his/her Bax genotype. The Bax genotype may be used alone orin conjunction with other genetic markers to conclusively identify anindividual or to rule out the individual as a possible perpetrator.

In one embodiment, human genomic DNA samples from a population ofindividuals (typically at least 50 persons from various racial origins)are individually aliquoted into reaction vessels (e.g., a well on amicrotitre plate). Each aliquot is digested (incubated) with one or morerestriction enzymes (e.g., EcoRI, HindIII, SmaI, BamHI, SalI, NotI,AccI, ApaI, BglII, XbaI, PstI) under suitable reaction conditions (e.g.,see New England Biolabs 1993 catalog). Corresponding digestion productsfrom each individual are loaded separately on an electrophoretic gel(typically agarose), electrophoresed, blotted to a membrane by Southernblotting, and hybridized with a labeled Bax probe (e.g., a full-lengthhuman Bax cDNA sequence of FIG. 3 or FIGS. 5 and 6). Restrictionfragments (bands) which are polymorphic among members of the populationare used as a basis to discriminate Bax genotypes and thereby classifyindividuals on the basis of their Bax genotype.

Similar categorization of Bax genotypes may be performed by sequencingPCR amplification products from a population of individuals and usingsequence polymorphisms to identify alleles (genotypes), and therebyidentify or classify individuals.

Methods of Rational Drug Design

Bax and Bcl-2 polypeptides, especially those portions which form directcontacts in Bax/Bcl-2 heterodimers, can be used for rational drug designof candidate Bcl-2-modulating agents (e.g., antineoplastics andimmunomodulators). The substantially purified Bax/Bcl-2 heterodimers andthe identification of Bax as a docking partner for Bcl-2 as providedherein permits production of substantially pure Bax/Bcl-2 polypeptidecomplexes and computational models which can be used for protein X-raycrystallography or other structure analysis methods, such as the DOCKprogram (Kuntz et al (1982) J. Mol. Biol. 161: 269; Kuntz I D (1992)Science 257: 1078) and variants thereof. Potential therapeutic drugs maybe designed rationally on the basis of structural information thusprovided. In one embodiment, such drugs are designed to preventformation of a Bax polypeptide: Bcl-2 polypeptide complex. Thus, thepresent invention may be used to design drugs, including drugs with acapacity to inhibit binding of Bax to Bcl-2.

The following examples are given to illustrate the invention, but arenot to be limiting thereof. All percentages given throughout thespecification are based upon weight unless otherwise indicated. Allprotein molecular weights are based on mean average molecular weightsunless otherwise indicated.

The nomenclature used hereafter and the laboratory procedures in cellculture, molecular genetics, and nucleic acid chemistry andhybridization described below may involve well known and commonlyemployed procedures in the art. Standard techniques are used forrecombinant nucleic acid methods, polynucleotide synthesis, andmicrobial culture and transformation (e.g., electroporation,lipofection). The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see, generally, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference)which are provided throughout this document.

Oligonucleotides can be synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Methods for PCR amplification are described in the art (PCR Technology:Principles and Applications for DNA Amplification ed. H A Erlich,Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guide to Methodsand Applications, eds. Innis, Gelfland, Snisky, and White, AcademicPress, San Diego, Calif. (1990); Mattila et al. (1991) Nucleic AcidsRes. 19: 4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods andApplications 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; and U.S. Pat. No. 4,683,202, which are incorporated herein byreference).

The experimental procedures, reagents, starting materials and testprocedures performed herein used the following techniques.

A) Cell Culture

RL7, a human B cell line which bears the t(14;18) and expresses highlevels of Bcl-2 was maintained in Iscove's modified Dulbecco's mediumsupplemented with 10% fetal calf serum (FCS)(supplied by Gibco). Theinterleukin-3 (IL-3) dependent murine cell line FL5.12, a lymphoidprogenitor clone, and all its derivatives were maintained in Iscove'smodified Dulbecco's medium supplemented with 10% FCS and 10% WEHI-3Bconditional medium as a source of IL-3.

B) Antibodies

The 6C8, human Bcl-2 specific hamster moAb (Hockenbery et al., 1990);the 12CA5, influenza virus hemagglutinin protein epitope specific murinemoAb (Kolodziej and Young, 1991); the 3F11, murine Bcl-2 specifichamster moAb (Veis et al, 1993) were used. 124, a human Bcl-2 specificmurine moAb was purchased from DAKO. TN3 19.12, a human TNF specifichamster moAb (Sheehan et al., 1989), was used as a control antibody. ForWestern immunostaining, the primary antibody dilutions were: 6C8(1:100), 3F11 (1:100), 12CA5 (1:50). The 3F11 antibody was directlybiotinylated, as described (Veis et al., 1993) for immunoblots. Theother antibodies were detected with species specific biotinylatedsecondary antibodies.

C) Immunoprecipitation and Western Blotting

Prior to metabolic labeling, cells were washed once in prewarmed,serum-free, methionine-free Dulbecco's medium. Cells were resuspended at3-5×10⁶ cells/ml in methionine-free Dulbecco's medium supplemented with10% dialyzed FCS and either 5% complete medium or 5% WEHI 3Bsupernatant. Metabolic labeling was performed with 40 uCi/ml of (³⁵S)methionine, (³⁵ S)cysteine for 9-12 hours before lysis. All steps ofthe immunoprecipitation were carried out on ice or in the cold room.Cells were washed twice with cold phosphate-buffered saline and lysed inan NP-40 isotonic lysis buffer with freshly added protease inhibitors(142.5 mM KCl, 5 mM MgCl2, 10 mM HEPES pH:7.2, 1 mM EGTA, 0.2% NP-40,0.2 mM phenylmethylsulfonyl fluoride, 0.1% Aprotinin, 0.7 ug/mlPepstatin and 1 ug/ml Leupeptin) by nutation for 30'. Nuclei and unlysedcellular debris were removed by centrifugation at 215,000×g for 10'.Lysates were precleared with 10% v/v Protein A-Sepharose (Prot.A-S) for30' which was removed by centrifugation at 400×g for 2'. In experiments,the lysates were also mixed with 10× excess cold cells lysates for 15'prior to the addition of the antibody. Specific antibodies were addedfor 90' and immunoprecipitates were captured with 10% v/v Prot.A-S for60'. In some experiments, supernatants of the primaryimmunoprecipitation were pre-cleared again with 10% v/v Prot.A-S for30', and re-precipitated with an appropriate second antibody.Immunoprecipitates were washed (unless indicated otherwise) once inlysis buffer followed by a wash in lysis buffer without NP-40.Immunoprecipitates were solubilized with SDS-PAGE sample buffer andelectrophoresed through 12.5% SDS-polyacrylamide gels. Gels containing(³⁵ S)methionine labeled proteins were fixed with 10% glacial aceticacid and 30% methanol overnight, enhanced by impregnating with acommercial fluorography enhancing solution (Enhance by DuPont) for 60',and precipitated in water for 30'. Gels were then dried andautoradiography performed at -70° C.

For immunoblots, proteins were electrotransferred overnight at 4° C. onnitrocellulose membranes. Filters were blocked for 2 hours withphosphate-buffered saline containing 3% non-fat milk. All additionalimmunostaining steps were performed in phosphate-buffered saline with0.05% Tween-20 (PBS-Tween) at room temperature. Filters were incubatedwith primary antibody for 2 hours. Species specific biotinylatedsecondary antibodies (1:300) were also reacted for 2 hours. Immunoblotswere reacted with horseradish-peroxidase-Streptavidin (1:1000) for 1hour. Filters were washed in PBX-Tween 4 times for 5' between each stepand were developed with diazobenzidine (BioRad) enhanced with nickelchloride (0.03%).

D) Peptide Sequencing

For protein isolation 1×10⁹ RL-7 or FL5.12-hBcl-2 cells wereimmunoprecipitated in large scale preparations, as described above.Immunoprecipitates were electrophoresed through a 3mm thick preparative12.5% SDS-polyacrylamide gel, stained with Coomassie blue dye for 10'and destained for 20'. Appropriate protein bands were excised from thegel and partially digested with an optimized amount of S. aureus V8protease (Calbiochem) "in gel", as described (Cleveland et al, 1976).Following separation on a second 17.5% SDS-polyacrylamide gel, resultingpeptide fragments were electrotransferred to a polyvinylidine difluoride(immobilon PVDF by Millipore) membrane. Filters were stained with 0.1%w/v Coomassie blue dye in 50% methanol for 7', destained with 50%methanol, 10% glacial acetic acid for 5', rinsed several times in waterand dried. Stained peptide fragments were excised and stored in amacrophage tube at 20° C. until microsequencing was performed by directN-terminal Edman degradation (Matsudaira, 1987).

For the cyanogen-bromide (CNBr) and o-phthaldehyde (OPA) protocol,large-scale immunoprecipitates were electrophoresed through a 3 mm thickpreparative 12.5% SDS-polyacrylamide gel, electrotransferred to PVDFmembrane and Coomassie dye stained, as above. p21 bands were excised,digested with CNBr at the methionine residues and sequenced. In certainsamples, amino acid ends were blocked with OPA at the cycle when anamino acid proline appeared (Hulmes et al., 1989). Sequencing resumedfrom the single peptide fragment that contained the unblocked imino acidproline at its N-terminus.

E) PCR Amplification and Cloning

Poly(A+) RNA from RL-7 cells was prepared by standard protocol primedwith oligo(dT) 15-mers and random hexamers (supplied from Pharmacia),and reverse transcribed with Moloney murine lymphotrophic virus reversetranscriptase (BRL) The generated complementary DNA (cDNA) was used in amixed oligonucleotide polymerase chain reactions (PCR) (Gould et al.,1989). Two mixed oligonucleotide pools, containing all possible codondegeneracies, were synthesized based on the determined amino acidsequence of human 21 kD Bax. The first primer pool was a mixture of 205617-mers corresponding to the amino acid sequence DPVPQD. The second(antisense) primer pool was derived from amino acid sequence IGDELD andwas a mixture of 2056 17-mers. A 100-ul PCR mixture contained 7890 pmolof each primer, 0.125 ug of cDNA, 2 mM of each DNTP, 10 mM Tris.HCL(pH:8.3), 50 mM KCl, 21.5 mM MgCl2, and 2.5 units of Thermus aquatics(Tag) DNA polymerase (supplied by Perkin-Elmer/Cetus). Thirty-eightamplification cycles consisted of: denaturation, 94° C. for 2' (firstcycle 4'); annealing, 60° C. 2'; extension, 72° C. 10". (last cycle60"). PCR products were size fractionated by agarose electrophoresis andthe expected 71 bp product was purified and directly ligated into a PCRcloning vector (TA cloning system by Invitrogen). Colonies containinginserts were selected and the insert sequenced with Sequenase (suppliedby United States Biochemical) using primers to the T7 and SP6 regions ofthe plasmid vector.

F) Screening of cDNA and Genomic Libraries

Standard techniques of molecular cloning were used as described, unlessindicated otherwise. Restriction enzymes were from Boehringer MannheimBiochemicals and New England Biolabs. The 712 bp PCR cDNA (FIG. 3, bps142-212) was radiolabeled by PCR and used to screen an Epstein-Barrvirus transformed human mature B cell (Namalwa) cDNA library (Ambrus etal., 1993) in lambda-ZAP II (supplied by Stratagene) by standardhybridization and washing at 2×SSC/0.1% SDS, 50° C. Three independentpositive clones were in vivo excised and sequenced. The longest of thesequenced inserts (820 bp) served as a probe to further screen theNamalwa human cDNA library, a human t(17;19) ALL early B cell (UOC-B1)cDNA library in lambda-ZAP II (Inaba et al., 1992) or an oligo(T)primed, size selected murine 70Z/3 pre-β cell cDNA library in lambdagt11 (Ben-Neriah, 1986). Several additional human and murine cDNA cloneswere obtained, subcloned, and sequenced. The final Bax sequences weredetermined on both strands.

For genomic screenings, a 129 SV murine genomic library in lambda FIX II(obtained from Stratagene), was screened with a full length murine BaxcDNA clone. The plaque purified genomic clones, that reacted with probesfor the 5' and 3' end of the cDNA, were selected. The insert of phageclone F1, that possesses the entire 5' through 3' ends of the cDNAsequence, was subcloned into Bluescript. Exon positions were placed byrestriction analysis and DNA sequencing defined the exon/intronboundaries.

G) Northern Analysis

Poly (A+) RNA from RL-7 and FL5.12 cells was prepared, electrophoresedin a denaturing 1.2% agarose-formaldehyde gel, and transferred onto anitrocellulose membrane. Filters were hybridized with various probes,and washed by standard protocol. However, for the exon 2 specific probe,the high stringency wash was in 2×SSC/0.1% SDS at 50° C.

H) Epitope Tagging and Expression Vector Construction

An eleven amino acid tag, that contained a well characterized epitope ofthe influenza virus hemagglutinin (HA) protein was attached to theN-terminus of murine Baxα (HA-Baxα) by a three step PCR approach(Kolodziej and Young, 1991). In the first, second and third rounds ofPCR, the ends of the Baxα ORF was stepwise extended by using 24-26 bpprimers in which the N-terminus possessed the epitope and the consensustranslation start site (Kozak, 1986), while the C-terminus had a stopcodon. In addition, an EcoRI site was also incorporated into each primerused in the third round of amplification. The PCR mixture contained 20pmol of each primer, 50 ng of cDNA, 2 mM of each DNTP, 10 mM Tris.HCL(pH:8.3), 50 mM KCl, 1.5 mM MgCl2, and 2.5 units of Thermus aquatics(Taq) DNA polymerase. Amplification of the target DNAs through 38 cycleswere: denaturation, 94° C. for 1'. (first cycle 4'.); annealing, 60° C.,1'. (first cycle 52° C.); extension, 72° C., 1'. (last cycle 10') PCRproducts were purified and either used as template for the next round ofamplification or directly ligated into a PCR cloning vector (TA cloningsystem by InVitrogen). The authenticity of the PCR reactions wereconfirmed by sequencing. The inserts were excised by EcoRI digestion,cloned into the EcoRI cloning site of SFFV-LTR-Neo expression vector(Fuhlbrigge et al, 1988) and transformed into competent XL-1 Blue cells(Strategene). Doubly CsCl purified and linearized sense-orientationplasmid constructs were transfected into FL5.12 cells by electroporation(200 V, 900 mF) with a BTX 300 transfector. Cells were recovered innonselective media for 24-48 hours, after which stably transfected cellswere selected for neomycin resistance in 2 mg/ml G418 (supplied byGibco), or for hygromycin resistance with 2 mg/ml hygromycin (suppliedby Calbiochem), by plating 4-12×10⁴ cells into 0.2 ml wells. Survivingclones were expanded and screened for the expression level of HA-Baxαand endogenous murine or transfected human Bcl-2 protein, bysolubilization and immunoblotting of equal amount of total protein. Highor low HA-Baxα and/or human Bcl-2 expressing clones were selected forsubsequent analysis.

I) Growth Factor Deprivation Studies

To assess the effect of Baxα on cell survival, stably transfected cellswere seeded at a concentration of 2×10⁵ cells/ml in the presence of IL-3for 24 hrs. Cells were washed thoroughly 3× in the serum free medium toremove the growth factor, and cultured at 5×10⁵ cells/ml in triplicate.Viabilities were determined at various time points by trypan blueexclusion counting at least 100 cells from each individual culture.

EXAMPLE 1

This Example demonstrates the co-precipitation of Bcl-2 with a 21 kDprotein.

Co-immunoprecipitation experiments were performed utilizing the 6C8monoclonal antibody (moAb) that is specific for human Bcl-2, and RL-7, ahuman B cell line which bears the t(14;18) translocation and expresseshigh levels of Bcl-2. A variety of detergent conditions were tested toidentify Bcl-2 associated proteins. When RL-7 cells were metabolicallylabeled with ³⁵ S-methionine, lysed and solubilized with 0.2% NP-40, anabundant 21 kD protein (p21) co-precipitated with Bcl-2. A lesser amountof a 24 kD species was also detected (FIG. 1).

In FIG. 1 cell lysates of (³⁵ S)methionine-labeled RL-7 cells wereimmunoprecipitated with an anti human Bcl-2 moAb in the presence (+) orabsence (-) of competitor cold lysate and washed as indicated above eachlane. When immunoprecipitates were washed in isotonic lysis solution theassociation remained intact. However, the addition of 0.1% SDS to thewash eliminated p21, indicating that p21 was a non-covalently associatedprotein rather than a Bcl-2 degradation product.

In the presence of excess cold RL-7 cell lysate radiolabeled p21 stillco-precipitated with Bcl-2 indicating that this was not anon-physiological association following cell lysis.

To confirm a specific interaction between Bcl-2 and p21, aninterleukin-3 (IL-3) dependent murine cell line, FL5.12 was examined. Inthe absence of IL-3, FL5.12 dies by apoptosis, but overexpression ofBcl-2 extends it survival.

Stable transfectants of FL5.12 expressing wild type human Bcl-2 (FL5.12hBcl-2) or a human Bcl-2 construct that lacks the COOH-terminalsignal-anchor sequence (Δ C-22) were generated. The Bcl-2 Δ C-22 proteinis no longer an integral membrane protein, yet it still provides partialprotection from cell death. Immunoprecipitation of human Bcl-2 with thespecies specific 6C8 moAb revealed an associated murine p21 protein inFL5.12 hBcl-2 cells (FIG. 2). In FIG. 2 cell lysates of (³⁵S)methionine-labeled FL5.12 clones transfected with vector only(Neo^(r)), with wild type human Bcl-2 (Bcl-2), or a deletion mutantlacking the signal-anchor sequence of human Bcl-2 (Δ C-22), wereimmunoprecipitated with an anti-human Bcl-2 moAb (6C8) or controlantibody (NS). The immunoprecipitates were analyzed by SDS-PAGE. The p21molecule was not detected when FL5.12 cells transfected with only aNeo^(R) vector were immunoprecipitated with 6C8. Similarly, an isotypematched control antibody, TN3 19.12 (NS) did not recognize the p21molecule in FL5.12-hBcl-2 cells. The Δ C-22 cytosolic form of Bcl-2 alsoco-precipitated mouse p21 from 0.2% NP-40 lysates, even though somewhatless efficiently. These findings demonstrate the specificity and theconservation of the interaction between p21 and Bcl-2 across species.For this reason we refer to p21 as Bax (ie. Bcl-2 Associated X protein).

EXAMPLE 2

This Example determined whether the induction of programmed cell deathaltered the association of Bcl-2 with Bax.

Immunoprecipitations were performed 12 hours following IL-3 withdrawal,a time point when FL5.12 cells begin to die and after which re-additionof IL-3 will not rescue cells. There was no change in the amount of Baxassociated with wild type or truncated Bcl-2 following IL-3 deprivation.

EXAMPLE 3

This Example demonstrates the molecular cloning of Bax.

To determine the identity of Bax, large scale 6C8 moAbimmunoprecipitates of human RL-7 and murine FL5.12-hBcl-2 cells wereelectrophoresed through a preparative SDS-polyacrylamide gel, andelectroblotted to polyvinylidine difluoride (PVFD) membrane. The p21(Bax) containing band was processed for microsequencing and theN-terminus proved to be blocked. Consequently internal peptide fragmentswere generated by V8 protease, cyanogen bromide (CNBr), or CNBr followedby 0-phthaldehyde in situ digestions. Peptide fragments were sequencedby Edman degradation and 2 overlapping internal fragments provided 29amino acids of human or murine sequences (FIG. 3).

In FIG. 3 the DNA sequence of the coding strand of human Bax andpredicted amino acid sequence is shown. Only those residues of themurine protein that are divergent are shown. The heavy underlined aminoacid residues correspond to the sequenced peptides of human (top) andmurine (bottom) Bax. The thin underlined amino acids correspond toresidues whose positions were unambiguously determined by aligning thecDNA sequence with the amino acid residues obtained from the sequencingof a mixture of peptide fragments generated by cyanogen bromidedigestion. The zig-zagged line marks the predicted transmembrane domainof Bax. Exon boundaries are denoted by numbers above the cDNA sequence.Arrows indicate the origin of degenerate primers used in themixed-oligonucleotide PCR amplification. The start of divergence betweenthe α and β form of Bax is indicated by the *.

The sequences isolated were not identical nor homologous to knownproteins and provide a basis for the cloning of the Bax cDNA. Twodegenerate primers (FIG. 3, arrows) corresponding to the DPVPQD (sense)and IDGELD (anti-sense) amino acid regions of the sequenced humanfragment were used in a mixed oligonucleotide polymerase chain reaction(PCR) with RL-7 cDNA as template. The predicted size 71 bp PCR productwas subcloned, its authenticity was verified by DNA sequencing, and itwas used as a probe to screen a human B cell cDNA library. An 820 bppartial cDNA clone was obtained, sequenced and used to screen additionalhuman and murine cDNA libraries. Multiple human and murine cDNA cloneswere obtained, subcloned and sequenced to establish the complete aminoacid sequence of Bax. To verify the authenticity of the predictedprotein deduced from the cDNA sequence, the amino acid residues obtainedby Edman degradation were aligned. FIG. 3 displays the human cDNAsequence and the deduced as well as direct amino acid sequence of bothhuman and murine Bax. All sequenced amino acids were accounted for andwere identical with the predicted protein sequence from the cDNAs.

The open reading frames (oRF) of both human and murine Bax are 576 bpand are 89.4% identical to one another. Both ORF encode a 192 amino acidprotein with a predicted molecular weight of 21.4 kD. The methionineinitiation codon of both the murine and human cDNA conforms to a Kozakconsensus sequence (Kozak, 1986). The murine and human Bax proteins arehighly conserved being 96% homologous with only six conservative andeight non-conservative amino acid changes, mostly in the N-terminalhalf. Both proteins have seven Ser/Thr residues that may represent sitesof phosphorylation. Hydropathicity analysis (Eisenberg et al., 1984)predicts the presence of a C-terminal transmembrane domain suggestingthat Bax exists as an integral membrane protein.

EXAMPLE 4

This Example demonstrates the genomic organization of the Bax gene.

Murine genomic clones were isolated by screening a genomic phage librarywith a murine Bax cDNA probe. Clones were plaque purified andcharacterized by restriction analysis. The location of exons and theexon-intron boundaries were determined by restriction enzyme analysisand genomic sequencing. The direction of the transcription is from leftto right.

Phage clone F1 contained a 16 kb genomic insert possessing the entire 5'through 3' ends of the cDNA sequence. The exon-intron boundaries weresequenced with primers derived from the cDNA and in all cases were inagreement with consensus exon/intron splice sequence requirements. TheBax gene consists of six exons all within a 4.5 kb region. Proteinencoding information is contributed by all six exons.

EXAMPLE 5

This Example demonstrates the alternative transcripts and tissuedistribution of the Bax gene.

The 70-Z/3 murine pre-B cell cDNA library consistently yielded clones ofa single Bax species whose sequence is shown in FIG. 3. However, aNamalwa human mature B cell and a t(17/19) early B leukemia cell(UOC-B1) cDNA library also yielded several clones that diverged from thesequence depicted in FIG. 3. These libraries possessed three species ofBax cDNAs shown in FIG. 4.

In FIG. 4, the boxes indicate exons identified by numbers. The shadingdifference between exon 3 for its RNA versus protein product in Bax γindicates a frameshift in exon 3 due to the alternative splicing of exon2. The ˜1.0 kb αRNA encodes the 192 amino acid 21 kD protein with thepredicted transmembrane segment A ˜1.5 kb βRNA encodes a 218 amino acid24 kD protein that lacks a hydrophobic terminus and may be a cytosolicform. The βRNA possesses an unspliced intron 5 of 630 bp accounting forthe apparent size increment from 1.0 to 1.5 kb (FIG. 4 and FIG. 5). InFIG. 5 the DNA sequence and predicted amino acid sequence code of theBaxβ form starting at the exon 5-intron 5 border. Intron 5 contributes60 amino acids before encountering a stop codon and lacks atransmembrane domain. The multiple cDNAs which represent the γ form ofRNA lack the small 53 bp exon 2 (FIG. 4). Both 1.0 kb and 1.5 kb formsof the γ RNA species were noted and are distinguished by the alternativesplicing of intron 5 (FIG. 4). The elimination of exon 2 shifts thereading frame in exon 3 which would contribute 30 novel amino acidsbefore encountering a stop codon (FIG. 6). In FIG. 6, the DNA sequenceand predicted amino acid sequence of the Bax γ form is shown. Iftranslated the γ RNAs would predict a protein of only 41 amino acidswith a molecular weight of 4.5 kd (FIG. 4).

To test if the predicted Bax species existed as mature mRNAs withincells, Northern blot analysis of poly (A+) RNA from FL5.12 and RL-7 wasperformed. An exon 1,3,4,5,6 containing cDNA probe identified a single1.0 kb RNA species in FL5.212 but a 1.0 and 1.5 kb RNA within RL-7. Boththe 1.0 and 1.5 kb RNAs were detected by an exon 6 specific probe, onlythe 1.5 kb RNA was identified. An exon 2 specific probe appeared tohybridize more strongly to the RNAs of the IL-3 dependent FL5.12 cellthan the immortalized RL-7 cell line. Thus, evidence exists by Northernand cDNA analysis for the existence of alternatively spliced α, β, and γRNA species that predict 3 types of proteins. FL5.12 cells possess the1.0 kb α RNA, and demonstrated only the p21 molecule in association withBcl-2 (FIG. 2). Of potential relationship, RL-7 revealed both a 1.0 kband α 1.5 kb β RNA and displayed both a p21 and p24 molecule associatedwith Bcl-2 (FIG. 1). It further appears that the Bcl-2 associated p24molecule in RL-7 is the product of the 1.5 kb Bax β RNA.

Northern analysis of total RNA from a survey of organs indicated thatBax was not lymphoid restricted but was widely expressed in a variety oftissues. Many tissues including lung, stomach, kidney and spleenexpressed both 1.0 and 1.5 kb RNA species. The 1.0 kb RNA was somewhatpreferential in heart and smooth muscle, whereas the duodenum revealedprincipally the 1.5 kb RNA. The 1.0 kb RNA was most abundant in thepancreas, while liver did not express substantial amounts of the gene.curiously the brain apparently possesses the 1.5 kb RNA and a highermolecular weight species. Northern analysis indicates a wide expressionof Bax and a splicing pattern that varies between lineages and celltypes.

EXAMPLE 6

This Example demonstrates that the Bax protein is homologous to Bcl-2.

The GenBank database when searched with the BLAST and TFASTA algorithmsrevealed a 20.8% identity and 43.2% similarity between p21 Bax and Bcl-2(FIG. 7). The alignment was maximized by introducing insertions markedby minuses. Identity across all 4 proteins is denoted in black shading,conservative changes are stippled, and the exon boundaries are numbered.The two most conserved regions, domain I and domain II, are boxed. Whilesome homology exists throughout the molecules the regions correspondingto exon 4 and 5 of Bax are the most conserved. The most highly conservedareas between Bcl-2 and Bax are denoted in FIG. 7 as domain I and domainII. Domain I is located on Bax exon 4, domain II on exon 5 and theputative transmembrane domain on exon 6. The juncture of Bax exon 5/6 atthe end of domain II is identical to the location of the exon 2/3juncture for Bcl-2. Of interest the retention of intron 5 at this siteresults in the β form of Bax RNA that lacks a transmembrane domain.Similarly, a β RNA form of Bcl-2 has been observed that lacks exon 3 andterminates in intron 2 predicting a β protein that would lack thesignal-anchor segment.

EXAMPLE 7

This Example demonstrates that overexpressed Bax α acceleratesprogrammed cell death.

The homology and physical association between Bax and Bcl-2 suggestedthat Bax might also modulate programmed cell death. Consequently, weoverexpressed Bax within FL5.12 cells which normally die by apoptosisfollowing withdrawal of IL-3. To follow the protein level of transfectedBax, an 11 amino acid tag containing a well characterized epitope of theinfluenza virus hemagglutinin (HA) protein was added to the N-terminusof murine Baxα by the PCR approach. This HA-Baxα insert was subclonedinto an expression construct utilizing the SFFV LTR as a constitutivelyexpressed promoter. FL5.12 cells were electroporated and G418 resistantstable clones were selected by limiting dilution. Clones were assessedfor levels of endogenous murine Bcl-2 with the 3F11 moAb specific formouse Bcl-2 and for Bax with the 12CA5 moAb specific for the HA epitopetag (FIGS. 8B, 8C).

A series of high and low Bax expressing clones were placed into IL-3deficient media and cell survival was monitored by vital dye exclusion.Neither overexpression of wild type Baxα or HA-Baxα has ever conferred asurvival advantage in the numerous clones that have been assessed.Instead, the presence of high levels of Bax have consistentlyaccelerated the rate of cell death FIG. 8A.

In FIG. 8A viability assays were performed. Triplicate cultures ofFL5.12 control cells (Neo^(r)), human Bcl-2 transfected (Bcl-2), andseveral independent clones constitutively expressing HA-Baxα weredeprived of IL-3. The percent viability was assessed by trypan blueexclusion as 12, 18, 24, 30, and 48 hrs following IL-3 deprivation andplotted as the mean ± standard error.

In FIGS. 8B and C, Western blot analysis of endogenous murine Bcl-2 (B)or HA-Baxα (C) protein in FL5.12 control cells (Neo) and HA-Baxα (cl.#)transfected FL5.12 clones. The murine Bcl-2 specific moAb 3F11 (B), orthe hemagglutinin epitope tag specific moAb 12CA5 (C) was used. HA-Baxαstably transfected clones 16, 18, 20, 22, and 23 and FL5.12 cellstransfected with a control vector lacking the Bax insert (Neo^(R))possessed comparable levels of endogenous murine Bcl-2 (FIG. 8B). Levelsof Baxα protein varied between very low (CL20), to high (CLs 16, 18, 22,23) (FIG. 8C). As shown in FIG. 8A Cl20 deviates only minimally from theNeo^(R) control cell line at 24-48 hours post IL-3 deprivation. However,high expressing CLs 16, 18, 22, and 23 display accelerated cell death at18-30 hrs and are nearly all dead at 48 hours compared to the 18%viability of the Neo^(R) line.

EXAMPLE 8

The Example demonstrates that the ratio of Bcl-2 to Bax affects the rateof programmed cell death.

To investigate the inter-relationship between levels of Bcl-2 and Bax weoverexpressed Bcl-2 in two cell lines with high levels of HA-Baxα.Clones 16 and 23 of the established HA-BAXα clones were co-transfectedwith a SFFV-hBcl-2 vector and an expression vector (LAP 267) providing ahygromycin selection marker. Hygromycin resistant clones were selectedand assessed for the expression of hBcl-2 with the 6C8 moAb and forlevels of HA-Baxα with the 12CA5 moAb (FIGS. 9B, C).

In FIG. 9A, viability assays were performed with triplicate cultures ofFL5.12 control cells (Neo^(r)), human Bcl-2 transfected (Bcl-2), andthree independent FL5.12 cells constitutively expressing both HA-Baxαand human Bcl-2 (Cls 16-8, 16-5, 23-5) were deprived of IL-3. Thepercent viability was assessed by trypan blue exclusion at severaltimepoints and plotted as the mean ± standard error. FIGS. 9B, C showWestern blot analysis of transfected human Bcl-2 (B) and HA-Baxα (C)protein in FL5.12 control cells (Neo), human Bcl-2 transfected (Bcl-2),and three independent FL5.12 cells constitutively expressing bothHA-Baxα and human Bcl-2 (CLs 16-5, 16-8, 23-5). The human Bcl-2 specificmoAb 6C8 (B) or the hemagglutinin epitope tag specific moAb 12CA5 wasused.

Clone 16-5 and 23-5 expressing high levels of hBcl-2 and Clone 16-8expressing intermediate amounts of hBcl-2 were selected for furtherstudy. Densitometry estimates of the relative amounts of the twoproteins within these cells revealed a relative ratio of hBcl-2/HA-Baxαof 2.04 for CL 16-5, 1.65 for CL 23-5, and 0.55 for Clone 16-8. The timecourse of apoptotic death following IL-3 deprivation was compared inthese clones and in FL5.12 cells possessing only Neo^(R) control vectoror only hBcl-2 (FIG. 9A). The addition of hBcl-2 partially countered theBax accelerated cell death. In multiple experiments the rate of celldeath paralleled the ratios of hBcl-2/HA-Baxα. Clones 16-5 and 23-5 ofthe double transfected clones demonstrated no death at 24 hrs whereasthe Neo^(R) control line was only 43% viable by this time. Clone 16-8with the lowest hBcl-2/HABaxα ratio was 73% viable at 24 hours but lostall viability by day 7. Clones 16-5 and 23-5 with high hBcl-2/HA-Baxαratios possessed viable cells over two weeks following IL-3 deprivation.Despite comparable levels of Bcl-2, Clone 16-5 and Clone 23-5 neverapproached the viability observed for the clone which only expressedhBcl-2 (FIG. 9A). Thus the presence of Bax also counters thedeath-repressor activity of Bcl-2.

EXAMPLE 9

This Example demonstrates that Bax forms homodimers and heterodimerizeswith Bcl-2.

The shared homology and reciprocal relationship between Bcl-2, Bax andcell survival prompted a further examination of their in vivoassociation. When HA-Baxα single transfected cells wereimmuno-precipitated with the HA tag specific 12CA5 moAb, a substantialamount of endogenous p21 Bax was co-precipitated (lane 5, FIG. 10A).Cell lysates of (³⁵ S)methionine-labeled FL5.12 clones transfected withhuman Bcl-2 (Bcl-2), with HA tagged murine Bax (Bax), or with both humanBcl-2 and HA tagged Bax (B+B), were immunoprecipitated with an antihuman Bcl-2 moAb (6C8), with an HA specific moAb (12CA5), or with amurine Bcl-2 specific moAb (3F11). Immunoprecipitated proteins wereresolved by SDS-PAGE. In FIG. 11 Western Blot analysis was performed ofprimary immunoprecipitates for murine Bcl-2 with a biotinylated 3F11moAb (left) or for transfected human Bcl-2 with the 6C8 moAb (right).FL5.12 control cells (Neo^(r)) or clones transfected with HA BAXα (Bax)or human Bcl-2 plus HA-Baxα (B+B) were immunoprecipitated with themurine Bcl-2 specific moAb (3F11), the HA specific moAb (12CA5), or witha human Bcl-2 specific moAb (124).

In FIG. 12 secondary immunoprecipitations of the supernatants from theprecipitates in FIG. 10 were performed. Designations are identical toFIG. 10. MoAbs used in the primary immunoprecipitations are shown inparenthesis. MoAbs used in the secondary immunoprecipitation followed.Immunoprecipitated proteins were revolved by SDS-PAGE.

In addition a very small amount of endogenous murine Bcl-2 was alsoprecipitated with HA-Baxα. Western blots of the 12CA5 moAb precipitatesconfirmed the identity of murine Bcl-2 (lane 1, FIG. 11).Immunoprecipitation of that remaining supernatant with the murine Bcl-2specific 3F11 moAb revealed that the majority of endogenous Bcl-2 didnot associate with Bax (lane 4, FIG. 12). This finding was confirmed byperforming the experiments in the reverse order. Most of the murineBcl-2 in a primary immunoprecipitate with the 3F11 moAb was notcomplexed with HA-Baxα or endogenous Bax (lane 6, FIG. 10). Yet, asecondary immunoprecipitation of that remaining supernatant with the12CA5 moAb revealed the majority of HA-Baxα was complexed with theendogenous Bax protein (lane 5, FIG. 12). Immunoblots developed with abiotinylated 3F11 moAb confirmed that the amount of endogenous murineBcl-2 associated with HA-Baxα was small compared to the total murineBcl-2 (lanes 1, 2 FIG. 11).

However, high levels of Bcl-2 protein introduced by a Bcl-2 expressionconstruct changed the ratio of Bcl-2/Bax heterodimers vs. Baxhomodimers. When double transfected cells were immunoprecipitated with6C8 moAb both epitope tagged and endogenous Bax complexed with theoverexpressed hBcl-2 (lane 3, FIG. 10). A secondary immunoprecipitationof that remaining supernatant with the 12CA5 moAb revealed that some ofthe Bax was not complexed with Bcl-2 and formed homodimers instead (lane3, FIG. 12). In reciprocal experiments, 12CA5 MoAb immunoprecipitatesalso contained hBcl-2 (lane 4, FIG. 10). Substantial amounts of hBcl-2appeared to be independent of Bax (compare lanes 3,4, FIG. 10). However,immunoprecipitation with 12CA5 was not complete in that the remainingsupernatant when immunoprecipitated with the 6C8 moAb revealedendogenous Bax and HA-Baxα in association with Bcl-2 (lane 2, FIG. 12).Yet, the intensity of the bands argued that a portion of hBcl-2 wasindependent of Bax molecules (lane 2, FIG. 12). Immunoblots confirmedthat substantial amounts of hBcl-2 were in the primaryimmunoprecipitates of HA-Baxα (lane 4, FIG. 11).

These studies establish that Bax homodimerizes and that theoverexpression of Bcl-2 competes for Bax by heterodimerization (lanes2,3,4, FIG. 10). This is also depicted in FIG. 13 wherein all indicatedprotein associations may represent dimers or higher oligomers. FreeBcl-2 is drawn alternatively as monomer or homodimer since somehomodimerization of Bcl-2 has been noted.

The site-specific mutagenesis work of Bcl-2 and its implications forprotein-protein interaction and definitively regulating a decisionalstep in the commitment to death has also been unexpectedly discovered.This is more particularly described in the following figures.

In FIG. 14, the cloning of the Bax cDNA established a family of Bcl-2closely related genes which were most highly conserved within segmentsknown as domain I and domain II. It is now clear that this is an evenwider family of molecules that includes Bcl-x, MCL-1 and two DNA virusproteins, LMW5-HL as well as BHRF-1 of the Epstein Barr Virus. As can beseen the homologies within these sets of proteins are focused withindomain I and II. Particularly dramatic is the middle segment of domain Iof FIG. 7, NWGR which is conserved all the way to the Epstein Barr virusprotein retaining the GR motif. The demonstration of the capacity of Baxand Bcl-2 to interact both in vivo and in vitro strongly indicates thatthese other Bcl-2 related proteins will also be involved inhomodimerization and heterodimerization with different members of thisfamily. Thus, all manipulations of domain I and domain II which areshown to affect the Bcl-2/Bax interaction and physiologic functions areequally applicable to all of the family members denoted.

FIG. 14B detailed the point mutations throughout the domain I in Bcl-2that were utilized. FIG. 14C denotes the tested mutations through theconserved domain II of FIG. 7.

FIG. 15A is an analysis of the level of Bcl-2 protein expression in theIL-3 dependent FL5.12 cell line that has been stably transfected withBcl-2 protein intracellularly as determined by flow cytometry. Asindicated the levels of the mutation products are comparable to that ofthe wild-type Bcl-2 clones. FIG. 15 is a Western blot of the same stabletransfectants of FL5.12 that confirm comparable levels of steady stateprotein. FIG. 15C is a parallel analysis of stable transfectants bearingthese expression constructs in the 2B4 T cell hybridoma that issensitive to dexamethasone as well as gamma irradiation induced death.Once again these reagents bear comparable levels of steady state Bcl-2protein. This figure establishes that any physiologic differences infunction that these molecules have is inherent to the altered pointmutations and not to quantitative levels of Bcl-2 protein.

FIG. 16 is an IL-3 deprivation time course of the stable transfects ofFL5.12. It demonstrates that wild-type Bcl-2 saves FL5.12 cells fromprogrammed cell death when compared to a control that has received aneomycin resistance expression vector only. Importantly, mutations indomain I that eliminate either the FRDG sequence or the WGR sequenceessentially eliminate the capacity of the Bcl-2 product to repressdeath. The rate of death returns to the same time course of the Neocontrol clone. Also noted is that a single amino acid alteration throughthe WGR sequence with a substitution of either an alanine in mI-3 or aglutamic acid within mI-4 for the glycine in the WGR sequence completelyeliminates the capacity of Bcl-2 to repress cell death. In fact thoseclones show an acceleration of death compared to the control. As we willdiscuss later this provides evidence that Bcl-2 may be interacting withother proteins beyond Bax.

FIG. 16B establishes that this death is an apoptotic programmed celldeath in which a nucleosomal length DNA degradation pattern is seen. InFIG. 16C a more quantitative measurement of this is provided looking atper cent of DNA fragmentation as a measurement of released DNA by adiphenylamine assay. This shows that the clones which express the mutantBcl-2 degrade and release their DNA in comparison to the wild-typeBcl-2. This essentially establishes the death pattern as beingapoptosis.

FIG. 17 shows a parallel study of viability in which the 2B4 T cellhybridoma bears either wild-type Bcl-2 or the WAR or WER mutant withindomain I. Bcl-2 wild-type confers resistance to either glucocorticoid orgamma irradiation induced programmed cell death. The presence ofmutations SM3 and SM4 eliminates Bcl-2's death repressor activity inthese signal transduction pathways of apoptosis as well. Once again theWER mutation shows an accelerated rate of cell death compared to theneomycin resistance containing control.

FIG. 18 shows immunoprecipitations of radiolabeled FL5.12 stabletransfectants in A and 2B4 stable transfectants in B.Immunoprecipitation of wild-type Bcl-2 always shows associated Bax andat times the p24 molecule. However, all of the mutations which disruptthe capacity for Bcl-2 to block death also disrupt its ability torecognize Bax.

FIG. 19 is a parallel assessment of stable transfectants of FL5.12 and2B4 cells with the mutations through domain II. The flow cytometryexamination of Bcl-2 protein levels indicate a comparable quantity ofthe mutant as compared to wild-type Bcl-2 in these stable clones. Panels19B, C and D confirm that at a Western blot level.

FIG. 20 tests the death responses of FL5.12 cells in A and 2B4 cells inC which bear the domain II mutants. The M3 and M5 mutations had noeffects upon the cell death pattern of either FL5.12 or 2B4. Thus, notall conserved amino acids within domain II mediate any functionaldifference in the Bcl-2 molecule. However, mutations of the QDN in theM2 set of mutants do disturb Bcl-2 function. However, the whole QDN hasto be eliminated in that the single substitution of M4 is not sufficientfor an effect. Panels B and D are the primary immunoprecipitants ofthese mutated molecules. They prove that the M2 mutations whichpartially destroy Bcl-2 function have a reduced association with Bax.However the M4 mutations which function normally has a full associationwith Bax. Thus, even changes in domain II which affect Bcl-2 functionappears to be mediated through its loss of interaction with the Baxmolecule.

In FIG. 21 cells are created that bear two expression constructs. TheDα15 cell line is an FL5.12 bearing human Bcl-2 wild-type and mouseBcl-2 wild-type vector. The DN2 bears a control Neomycin resistantexpression vector and mouse Bcl-2 wild-type vector. The clone DSM4-5contains mutant Bcl-2 of domain I and a mouse Bcl-2 wild-type whereasthe DSM-4 contains another Bcl-2 mutant of human origin with mouse Bcl-2wild-type. The flow of cytometry histograms in FIG. 21 denote that thedouble transfectants bear comparable levels of the mouse Bcl-2 wild-typeprotein. FIGS. 21B and C prove that the mutations of Bcl-2 thatinterfere with its capacity to associate with Bax still allow theformation of Bcl-2 homodimers. In FIG. 21B we see a primaryimmunoprecipitation of either wild-type human Bcl-2 or the mutated humanBcl-2 in which the 3-4 and 4-7 mutants have no associated Bax. Thoseimmunoprecipitations are then developed by Western blot analysis with ananti-mouse Bcl-2 antibody. Both the wild-type human Bcl-2 as well as thetwo mutants mI 3-4 and mI 4-7 both associate with the wild-type mouseBcl-2. Moreover, cross linking experiments indicate the capacity ofBcl-2 to homodimerize as well as heterodimerize. The Bcl-2 homodimersare seen with mI3-4 and mI4-7 as well.

The results of this data firmly establishes that agents that disrupt thecapacity of Bcl-2 to interact with Bax will eliminate the deathrepressor activity. Such cells are very vulnerable to programmed celldeath. Consequently any therapeutic strategy that eliminates thisprotein--protein heterodimerzation would be a fundamental andexceptionally successful mechanism to kill cells. Such an approach hasimportance for cancer therapy, the elimination of autoreactive cells inautoimmunity, and the elimination of hyperplasias in a variety ofpathologic hypertrophies such as benign prostatic hypertrophy,lymphoproliferative diseases and the like.

These have not only been created in vivo mammalian cell line systems asscreening reagents but we have also created an in vitro protein--proteinassociation assay as well as a yeast two hybrid system assay forscreening chemical compounds and synthetic peptides which would disruptBcl-2/Bax interactions. This approach is also being applied to thedisruption of interactions between all of these family members whichinteract through domain I and domain II.

An in vitro protein interaction system has been created in which Baxtagged with glutathione-S-transferase (GST) and attached to aglutathione bearing bead is interacted with a radiolabeled member ofthis family such as Bcl-2. In such a system Bcl-2 will associate withBax and can be precipitated with the bead. This provides a rapid invitro screening assay in which synthetic peptides which mimic the domainI and domain II structures can be screened for their capacity to disruptthe association of Bcl-2 and Bax and other related family members. Sucha system can also be utilized as a rapid through-put to screen selectedand random chemical libraries for their capacity to interfere with thisinteraction.

As a first level of in vivo interference of protein--proteinheterodimerization we have created a yeast 2 hybrid system. In thissystem the Bcl-2ΔC22 is fused to a ga14 DNA binding domain while theBaxΔC23 is fused to a ga14 activation domain. We have shown that Bcl-2and Bax interact, heterodimerizing within yeast cells, enabling theactivation of a lacZ reporter driven by a ga14 DNA recognition motif.This provides a rapid through-put easily quantifiable, simplespectrophotometry assay for therapeutic products that could result in adisruption of Bcl-2/Bax interactions. Successful molecules identified bythe in vitro protein--protein interaction or primarily isolated fromthis assay could be identified.

Ultimately, reagents identified in such systems could be confirmed to beof biologic importance in the mammalian cell lines established. Thoseinclude the FL5.12 and 2B4 clones bearing stable transfections of Bcl-2,Bax and their modified analogs. Ultimately, proof of concept on anytherapeutic agent could proceed to testing in our in vivo models inwhich we have transgenic mice overexpressing Bcl-2, or overexpressingBax.

The present invention thus find wide application to a multitude oftreatment regimens and diagnostic uses.

It may be used in any therapy which regulates the ratio of Bcl-2/Bax andwill alter the rheostat of a cell's selection of survival versus death.This is a powerful therapeutic modality in which changing the ratio tofavor Bax and cell death would be applicable to hyperplasias,hypertrophies, cancers and autoimmunity. Altering the ratio to promotethe survival of cells by having Bcl-2 in excess would be a successfulstrategy in the treatment of neuro-degenerative disease as well asimmunodeficiency, ischemia induced injury such as myocardial infarctionand neurologic stroke. This would include regulating either Bcl-2 or Baxat the gene transcription level or at the protein half-life or proteinmodification level.

In this regard, a method for modulating apoptosis of a cell, typically alymphocyte, is provided by this invention. The method comprisesadministering to a cell an agent which alters intermolecular bindingbetween Bcl-2 and Bax proteins, typically by inhibiting formation ofheteromultimers (e.g., heterodimers) between Bcl-2 and Bax and/orhomomultimers of Bcl-2 or Bax. Administration of such agents canselectively inhibit formation of Bax/Bax homodimers or Bax/Bcl-2heterodimers or higher multimeric forms having biological activity. Inone embodiment, the agent is a compound comprising a structure of a Baxprotein domain I or domain II polypeptide domain; for example, apolypeptide comprising a Bax domain I or domain II sequence can serve assuch an agent if deliverable intracellularly. In an embodiment, theagent is a compound comprising a structure of a Bcl-2 protein domain Ior domain II polypeptide domain comprising a sequence variation (e.g.,mutation) which reduces the agent's affinity for Bax and which does notsubstantially reduce the agent's affinity for Bcl-2, whereby the agentcompetitively inhibits formation of Bcl-2/Bcl-2 homodimers comprisingnaturally-occurring 3cl-2 but does not substantially inhibit formationof Bcl-2/Bax heterodimers or other heteromultimers.

In another aspect of the invention, the method(s) of modulatingapoptosis of a cell by administering an agent which altersintermolecular binding between Bcl-2 and Bax proteins are used to treata pathological condition in a patient. For example, a patient with apathological condition wherein abnormal cell proliferation or abnormalcell apoptosis is an underlying etiology may be treated by administeringan agent which modulates the amount of Bax protein present in cell(e.g., a neoplastic or hyperplastic cell) and/or the ratio of Bax:Bcl-2proteins in a cell and/or the ratios of Bax/Bax homomultimers, Bax/Bcl-2heteromultimers, and Bcl-2/Bcl-2 homomultimers.

In another aspect of the invention, an antisense polynucleotide isadministered to inhibit transcription and/or translation of Bax in acell.

In another aspect of the invention, a polynucleotide encoding a Baxpolypeptide is delivered to a cell, such as an explanted lymphocyte,hematopoietic stem cell, bone marrow cell, and the like. The deliveredpolynucleotide, typically including an operably-linked promoter (andoptionally enhancer) to drive transcription of the Bax-encodingpolynucleotide providing expression of a Bax polypeptide, is transferredto the cell to form a stably or transiently transfected cell orhomologous recombinant cell wherein Bax protein is expressed under thecontrol of a predetermined transcriptional control sequence. Suchtransfected cells may be transferred into a patient (e.g., the patientfrom which the cells were originally explanted) for therapy of adisease, such as a neoplastic disease, and may be used, in oneembodiment, to reconstitute hematopoietic cells followingchemotherapy/radiotherapy. Such methods may be used, for example, ingene therapy (e.g., to treat neoplasia, hyperplasia, autoimmunediseases, and the like) and Bax polynucleotides may be used inconjunction with suitable gene therapy modalities and delivery systems(e.g., adenoviral vectors and the like).

In another aspect of the invention, transgenic nonhuman animals, such asmice, bearing a transgene encoding a Bax polypeptide and/or a Bcl-2polypeptide are provided. Such transgenes may be homologously recombinedinto the host chromosome or may be non-homologously integrated.Typically, such transgenes comprise a sequence encoding a Baxpolypeptide (or Bcl-2 polypeptide) wherein the polynucleotide sequenceis operably linked to a transcription control sequence (e.g.,promoter/enhancer) for modulatable (e.g., inducible and/or repressible)or constitutive transcription of the Bax (or Bcl-2) encoding sequence.In one variation, the endogenous Bax gene is functionally disrupted bygene targeting via homologous recombination with a targeting construct.Nonhuman animals harboring such Bax functionally disrupted alleles(i.e., "gene knockouts"), generally homozygous for such Bax knockoutsmay also comprise a Bax transgene, such that Bax is expressed under thetranscriptional control of an operably linked transcriptional controlsequence other than the naturally occurring transcriptional controlsequence of the nonhuman animal's endogenous Bax gene.

The invention also provides host cells expressing Bax polypeptidesencoded by a polynucleotide other than a naturally-occurring Bax gene ofthe host cell. An exogenous polynucleotide sequence encoding a Baxpolypeptide or portion thereof can be transferred into a host cell andtranscribed under the control of a transcriptional control sequence suchthat a Bax polypeptide is expressed. In one variation, the Baxpolypeptide(s) can be recovered from the host cell alone or inconjunction with one or more other polypeptide species associated withit. Such host cells may further comprise a polynucleotide sequenceencoding a Bcl-2 polypeptide other than the naturally-occurring Bcl-2gene of the host cell; such cells may express a Bcl-2 polypeptide and aBax polypeptide; such cells may further comprise knockout alleles of Baxand/or Bcl-2. In one variation, the host cell is a yeast cell and theBax and/or Bcl-2 polypeptide is expressed as a fusion protein; oneembodiment of this variation employs a yeast two-hybrid expressionsystem.

The invention provides antibodies, both monoclonal antibodies andpolyclonal antisera, which specifically bind to a Bax polypeptide withan affinity of about at least 1×10⁷ M⁻¹, typically at least 1×10⁸ M⁻¹ ormore.

It may also be used in any therapy which disrupts the Bcl-2/Baxheterodimerization which would lead to the death of cells by theelimination of Bcl-2 death repressor activity. This would include randomscreens of chemicals, compounds that would be able to do that, as wellas peptides which would lead through molecular modeling to organicchemicals that would also disrupt this association.

In this regard, the invention provides screening assays for identifyingagents which modulate (e.g., inhibit) binding of a Bax polypeptide to aBcl-2 polypeptide and/or which modulate (e.g., inhibit) binding of a Baxpolypeptide to a Bax polypeptide. The compositions of such screeningassays generally comprise a Bax polypeptide and a Bcl-2 polypeptide in asuitable aqueous binding solution or in a cell (e.g., a yeast ormammalian cell, a bacterium, a plant cell); the Bax and Bcl-2polypeptides generally comprise a domain I sequence and/or a domain IIsequence. An agent is added to such a screening assay and the formationof Bax/Bcl-2 heteromultimers (heterodimers) is determined; agents whichreduce or argument the formation of Bax/Bcl-2 heteromultimers ascompared to a parallel control Bax/Bcl-2 binding reaction lacking theagent are thereby identified as Bax/Bcl-2 modulators. Optionally, oralternatively, the capacity of an agent to inhibit or argument formationof Bcl-2/Bcl-2 homomultimers (homodimers) and/or Bax/Bax homomultimers(homodimers) can be measured relative to a control binding reactionlacking the agent; such assays identify Bcl-2 modulators and/or Baxmodulators. Agents which selectively or preferentially inhibit Bax/Bcl-2heteromultimer (homodimer) formation as compared to Bax/Bax orBcl-2/Bcl-2 homomultimer (homodimer) formation can be identified by theassays.

In another aspect, candidate agents are identified by their ability toblock the binding of a Bax polypeptide to a Bcl-2 polypeptide. The Baxpolypeptide includes one or more Bcl-2 binding sites at which a Bcl-2protein specifically binds. One means for detecting binding of a Baxpolypeptide to a Bcl-2 polypeptide is to immobilize one of thepolypeptide species, such as by covalent or noncovalent chemical linkageto a solid support, and to contact the immobilized Bax (or Bcl-2)polypeptide with a Bcl-2 (or Bax) polypeptide that has been labeled witha detectable marker (e.g., by incorporation of radiolabeled amino acid).Such contacting is typically performed in aqueous conditions whichpermit binding of a Bax (or Bcl-2) polypeptide to a Bcl-2 (or Bax)polypeptide containing a binding sequence, such as domain I or domainII. Binding of the labeled Bax (or Bcl-2) to the immobilized Bcl-2 (orBax) is measured by determining the extent to which the labeledpolypeptide is immobilized as a result of a specific bindinginteraction. Such specific binding may be reversible, or may beoptionally irreversible if a cross-linking agent is added in appropriateexperimental conditions.

In a variation of the invention, polynucleotides of the invention areemployed for diagnosis of pathological conditions or genetic diseasethat involve neoplasia or abnormal apoptosis, and more specificallyconditions and diseases that involve alterations in the structure orabundance of Bax.

The invention also provides Bax polynucleotide probes for diagnosis ofpathological conditions (e.g., neoplasia, AIDS, hyperplasia, congenitalgenetic diseases) by detection of Bax mRNA or rearrangements oramplification of the Bax gene in cells explanted from a patient, ordetection of a pathognomonic Bax allele (e.g., by RFLP orallele-specific PCR analysis). Typically, the detection will be by insitu hybridization using a labeled (e.g., ³² P, ³⁵ S, ¹⁴ C, ³ H,fluorescent, biotinylated, digoxigeninylated) Bax polynucleotide,although Northern blotting, dot blotting, or solution hybridization onbulk RNA or poly A⁺ RNA isolated from a cell sample may be used, as mayPCR amplification using Bax-specific primers. Cells which contain anincreased or decreased amount or altered structure of Bax mRNA ascompared to cells of the same cell type(s) obtained from a normalundiseased control source will be identified as candidate pathologicalcells. Similarly, the detection of pathognomonic rearrangements oramplification of the Bax locus or closely linked loci in a cell samplewill identify the presence of a pathological condition or apredisposition to developing a pathological condition (e.g., cancer,genetic disease) and may be used for forensic identification ofindividual identity and paternity.

Polynucleotide sequences encoding Bax are also provided. Thecharacteristics of the cloned sequences are given, including thenucleotide and predicted amino acid sequence in FIGS. 3, 5 and 6.Polynucleotides comprising sequences encoding these amino acid sequencescan serve as templates for the recombinant expression of quantities ofBax polypeptides, such as human Bax and murine Bax. Polynucleotidescomprising such sequences can also serve as probes for nucleic acidhybridization to detect the transcription and mRNA abundance of Bax mRNAin individual lymphocytes (or other cell types) by in situhybridization, and in specific lymphocyte populations by Northern blotanalysis and/or by in situ hybridization (Alwine et al. (1977) Proc.Natl. Acad. Sci. U.S.A. 74: 5350) and/or PCR amplification and/or LCRdetection. Such recombinant polypeptides and nucleic acid hybridizationprobes can be used in conjunction with in vitro screening methods forpharmaceutical agents (e.g., antineoplastic agents, immunomodulators)and for diagnosis and treatment of neoplastic or preneoplasticpathological conditions, genetic diseases, and other pathologicalconditions.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Furthermore, the Bcl-2/Bax experimental system serves as a generalizableparadigm for the differential regulation of all molecules that repressor accelerate cell death. The alterations of their inherent ratios orthe disruption of their protein--protein interactions as eitherhomodimers or heterodimers reflects a powerful and predicted approachfrom these experimental data.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 31                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 624 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..624                                                #/note= "Human BAX cDNA"RMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GAGGCGGCGG CGGGAGCGGC GGTGATGGAC GGGTCCGGGG AGCAGCCCAG AG - #GCGGGGGG         60                                                                          - CCCACCAGCT CTGAGCAGAT CATGAAGACA GGGGCCCTTT TGCTTCAGGG TT - #TCATCCAG        120                                                                          - GATCGAGCAG GGCGAATGGG GGGGGAGGCA CCCGAGCTGG CCCTGGACCC GG - #TGCCTCAG        180                                                                          - GATGCGTCCA CCAAGAAGCT GAGCGAGTGT CTCAAGCGCA TCGGGGACGA AC - #TGGACAGT        240                                                                          - AACATGGAGC TGCAGAGGAT GATTGCCGCC GTGGACAGAG ACTCCCCCCG AG - #AGGTCTTT        300                                                                          - TTCCGAGTGG CAGCTGACAT GTTTTCTGAC GGCAACTTCA ACTGGGGCCG GG - #TTGTCGCC        360                                                                          - CTTTTCTACT TTGCCAGCAA ACTGGTGCTC AAGGCCCTGT GCACCAAGGT GC - #CGGAACTG        420                                                                          - ATCAGAACCA TCATGGGCTG GACATTGGAC TTCCTCCGGG AGCGGCTGTT GG - #GCTGGATC        480                                                                          - CAAGACCAGG GTGGTTGGGA CGGCCTCCTC TCCTACTTTG GGACGCCCAC GT - #GGCAGACC        540                                                                          - GTGACCATCT TTGTGGCGGG AGTGCTCACC GCCTCGCTCA CCATCTGGAA GA - #AGATGGGC        600                                                                          #               624TTGG ACTG                                                  - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 192 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..192                                                #/note= "Human BAX polypeptide":                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gl - #y Gly Pro Thr Ser Ser         #                15                                                           - Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Le - #u Gln Gly Phe Ile Gln         #            30                                                               - Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pr - #o Glu Leu Ala Leu Asp         #        45                                                                   - Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Le - #u Ser Glu Cys Leu Lys         #    60                                                                       - Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Gl - #u Leu Gln Arg Met Ile         #80                                                                           - Ala Ala Val Asp Arg Asp Ser Pro Arg Glu Va - #l Phe Phe Arg Val Ala         #                95                                                           - Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Tr - #p Gly Arg Val Val Ala         #           110                                                               - Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Ly - #s Ala Leu Cys Thr Lys         #       125                                                                   - Val Pro Glu Leu Ile Arg Thr Ile Met Gly Tr - #p Thr Leu Asp Phe Leu         #   140                                                                       - Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gl - #n Gly Gly Trp Asp Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gl - #n Thr Val Thr Ile Phe         #               175                                                           - Val Ala Gly Val Leu Thr Ala Ser Leu Thr Il - #e Trp Lys Lys Met Gly         #           190                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 192 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..192                                                #/note= "Murine BAX polypeptide"                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Asp Gly Ser Gly Glu Gln Leu Gly Ser Gl - #y Gly Pro Thr Ser Ser         #                15                                                           - Glu Gln Ile Met Lys Thr Gly Ala Phe Leu Le - #u Gln Gly Phe Ile Gln         #            30                                                               - Asp Arg Ala Gly Arg Met Ala Gly Glu Thr Pr - #o Glu Leu Thr Leu Glu         #        45                                                                   - Gln Pro Pro Gln Asp Ala Ser Thr Lys Lys Le - #u Ser Glu Cys Leu Arg         #    60                                                                       - Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Gl - #u Leu Gln Arg Met Ile         #80                                                                           - Ala Asp Val Asp Arg Asp Ser Pro Arg Glu Va - #l Phe Phe Arg Val Ala         #                95                                                           - Ala Asp Met Phe Ala Asp Gly Asn Phe Asn Tr - #p Gly Arg Val Val Ala         #           110                                                               - Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Ly - #s Ala Leu Cys Thr Lys         #       125                                                                   - Val Pro Glu Leu Ile Arg Thr Ile Met Gly Tr - #p Thr Leu Asp Phe Leu         #   140                                                                       - Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gl - #n Gly Gly Trp Glu Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gl - #n Thr Val Thr Ile Phe         #               175                                                           - Val Ala Gly Val Leu Thr Ala Ser Leu Thr Il - #e Trp Lys Lys Met Gly         #           190                                                               - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 186 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..186                                                #/note= "DNA sequence of BAX beta                                                            transcript"                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - TGGGTGAGAC TCCTCAAGCC TCCTCACCCC CACCACCGCG CCCTCACCAC CG - #CCCCTGCC         60                                                                          - CCACCGTCCC TGCCCCCCGC CACTCCTCTG GGACCCTGGG CCTTCTGGAG CA - #GGTCACAG        120                                                                          - TGGTGCCCTC TCCCCATCTT CAGATCATCA GATGTGGTCT ATAATGCGTT TT - #CCTTACGT        180                                                                          #          186                                                                - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 61 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..61                                                 #/note= "BAX polypeptide of gamma                                                            mRNA tran - #script"                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Trp Val Arg Leu Leu Lys Pro Pro His Pro Hi - #s His Arg Ala Leu Thr         #                15                                                           - Thr Ala Pro Ala Pro Pro Ser Leu Pro Pro Al - #a Thr Pro Leu Gly Pro         #            30                                                               - Trp Ala Phe Trp Ser Arg Ser Gln Trp Cys Pr - #o Leu Pro Ile Phe Arg         #        45                                                                   - Ser Ser Asp Val Val Tyr Asn Ala Phe Ser Le - #u Arg Val                     #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 126 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..126                                                #/note= "Human BAX DNA"ORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - ATGGACGGGT CCGGAGAGCA GCCCAGAGGC GGGGTTTCAT CCAGGATCGA GC - #AGGGCGAA         60                                                                          - TGGGGGGGGA GGCACCCGAG CTGGCCCTGG ACCCGGTGCC TCAGGATGCG TC - #CACCAAGA        120                                                                          #          126                                                                - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 41 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..41                                                 #/note= "Human BAX polypeptide":                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gl - #y Val Ser Ser Arg Ile         #                15                                                           - Glu Gln Gly Glu Trp Gly Gly Arg His Pro Se - #r Trp Pro Trp Thr Arg         #            30                                                               - Cys Leu Arg Met Arg Pro Pro Arg Ser                                         #        40                                                                   - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 192 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..192                                                #/note= "Murine BAX polypeptide"                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Asp Gly Ser Gly Glu Gln Leu Gly Ser Gl - #y Gly Pro Thr Ser Ser         #                15                                                           - Glu Gln Ile Met Lys Thr Gly Ala Phe Leu Le - #u Gln Gly Phe Ile Gln         #            30                                                               - Asp Arg Ala Gly Arg Met Ala Gly Glu Thr Pr - #o Glu Leu Thr Leu Glu         #        45                                                                   - Gln Pro Pro Gln Asp Ala Ser Thr Lys Lys Le - #u Ser Glu Cys Leu Arg         #    60                                                                       - Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Gl - #u Leu Gln Arg Met Ile         #80                                                                           - Ala Asp Val Asp Thr Asp Ser Pro Arg Glu Va - #l Phe Phe Arg Val Ala         #                95                                                           - Ala Asp Met Phe Ala Asp Gly Asn Phe Asn Tr - #p Gly Arg Val Val Ala         #           110                                                               - Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Ly - #s Ala Leu Cys Thr Lys         #       125                                                                   - Val Pro Glu Leu Ile Arg Thr Ile Met Gly Tr - #p Thr Leu Asp Phe Leu         #   140                                                                       - Arg Glu Arg Leu Leu Val Trp Ile Gln Asp Gl - #n Gly Gly Trp Glu Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gl - #n Thr Val Thr Ile Phe         #               175                                                           - Val Ala Gly Val Leu Thr Ala Ser Leu Thr Il - #e Trp Lys Lys Met Gly         #           190                                                               - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 192 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..192                                                #/note= "Human BAX polypeptide":                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gl - #y Gly Pro Thr Ser Ser         #                15                                                           - Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Le - #u Gln Gly Phe Ile Gln         #            30                                                               - Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pr - #o Glu Leu Ala Leu Asp         #        45                                                                   - Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Le - #u Ser Glu Cys Leu Lys         #    60                                                                       - Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Gl - #u Leu Gln Arg Met Ile         #80                                                                           - Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Va - #l Phe Phe Arg Val Ala         #                95                                                           - Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Tr - #p Gly Arg Val Val Ala         #           110                                                               - Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Ly - #s Ala Leu Cys Thr Lys         #       125                                                                   - Val Pro Glu Leu Ile Arg Thr Ile Met Gly Tr - #p Thr Leu Asp Phe Leu         #   140                                                                       - Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gl - #n Gly Gly Trp Asp Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gl - #n Thr Val Thr Ile Phe         #               175                                                           - Val Ala Gly Val Leu Thr Ala Ser Leu Thr Il - #e Trp Lys Lys Met Gly         #           190                                                               - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 239 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..239                                                #/note= "Human Bcl-2 polypeptide"                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Ala His Ala Gly Arg Thr Gly Tyr Asp As - #n Arg Glu Ile Val Met         #                15                                                           - Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gl - #y Tyr Glu Trp Asp Ala         #            30                                                               - Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Al - #a Pro Ala Pro Gly Ile         #        45                                                                   - Phe Ser Ser Gln Pro Gly His Thr Pro His Pr - #o Ala Ala Ser Arg Asp         #    60                                                                       - Pro Val Ala Arg Thr Ser Pro Leu Gln Thr Pr - #o Ala Ala Pro Gly Ala         #80                                                                           - Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pr - #o Val Val His Leu Thr         #                95                                                           - Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg Ar - #g Tyr Arg Arg Asp Phe         #           110                                                               - Ala Glu Met Ser Ser Gln Leu His Leu Thr Pr - #o Phe Thr Ala Arg Gly         #       125                                                                   - Arg Phe Ala Thr Val Val Glu Glu Leu Phe Ar - #g Asp Gly Val Asn Trp         #   140                                                                       - Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gl - #y Val Met Cys Val Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Val Asn Arg Glu Met Ser Pro Leu Val As - #p Asn Ile Ala Leu Trp         #               175                                                           - Met Thr Glu Tyr Leu Asn Arg His Leu His Th - #r Trp Ile Gln Asp Asn         #           190                                                               - Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gl - #y Pro Ser Met Arg Pro         #       205                                                                   - Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Th - #r Leu Leu Ser Leu Ala         #   220                                                                       - Leu Val Gly Ala Cys Ile Thr Leu Gly Ala Ty - #r Leu Gly His Lys             225                 2 - #30                 2 - #35                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 236 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..236                                                #/note= "Murine Bcl-2 polypeptide"                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Met Ala Gln Ala Gly Arg Thr Gly Tyr Asp As - #n Arg Glu Ile Val Met         #                15                                                           - Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gl - #y Tyr Glu Trp Asp Ala         #            30                                                               - Gly Asp Ala Asp Ala Ala Pro Leu Gly Ala Al - #a Pro Thr Pro Gly Ile         #        45                                                                   - Phe Ser Phe Gln Pro Glu Ser Asn Pro Met Pr - #o Ala Val His Arg Glu         #    60                                                                       - Met Ala Ala Arg Thr Ser Pro Leu Arg Pro Le - #u Val Ala Thr Ala Gly         #80                                                                           - Pro Ala Leu Ser Pro Val Pro Pro Cys Val Hi - #s Leu Thr Leu Arg Arg         #                95                                                           - Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Ar - #g Asp Phe Ala Glu Met         #           110                                                               - Ser Ser Gln Leu His Leu Thr Pro Phe Thr Al - #a Arg Gly Arg Phe Ala         #       125                                                                   - Thr Val Val Glu Glu Leu Phe Arg Asp Gly Va - #l Asn Trp Gly Arg Ile         #   140                                                                       - Val Ala Phe Phe Glu Phe Gly Gly Val Met Cy - #s Val Glu Ser Val Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Glu Met Ser Pro Leu Val Asp Asn Ile Al - #a Leu Trp Met Thr Glu         #               175                                                           - Tyr Leu Asn Arg His Leu His Thr Trp Ile Gl - #n Asp Asn Gly Gly Trp         #           190                                                               - Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Me - #t Arg Pro Leu Phe Asp         #       205                                                                   - Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Se - #r Leu Pro Trp Val Gly         #   220                                                                       - Ala Cys Ile Thr Leu Gly Ala Tyr Leu Gly Hi - #s Lys                         225                 2 - #30                 2 - #35                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..21                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Glu Glu Leu Phe Phe Arg Asp Gly Val Asn Tr - #p Gly Arg Ile Val Ala         #                15                                                           - Phe Phe Glu Gly Gly                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..23                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Ala Asp Met Phe Phe Ser Asp Gly Asn Phe As - #n Trp Gly Arg Val Val         #                15                                                           - Ala Leu Phe Tyr Phe Ala Ser                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..23                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Ile His Val Phe Phe Ser Asp Gly Val Thr As - #n Trp Gly Arg Ile Val         #                15                                                           - Thr Leu Ile Ser Phe Gly Ala                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Region                                                          (B) LOCATION: 5                                                     #/note= "Amino acid is either K:                                                        (Lys) or R (Arg)"                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..21                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Thr Glu Leu Phe Xaa Asp Leu Ile Asn Trp Gl - #y Arg Ile Cys Gly Phe         #                15                                                           - Ile Val Phe Ser Ala                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..21                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Leu Glu Ile Phe His Arg Gly Asp Pro Ser Le - #u Gly Arg Ala Leu Ala         #                15                                                           - Trp Met Ala Cys Met                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..23                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Arg His Leu His Thr Trp Thr Gln Asp Asn Gl - #y Gly Trp Asp Ala Phe         #                15                                                           - Val Glu Leu Tyr Gly Pro Ser                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..23                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gl - #y Gly Trp Asp Leu Leu         #                15                                                           - Ser Gly Tyr Phe Gly Thr Pro                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..22                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Arg Thr Lys Arg Asp Trp Leu Val Lys Gln Ar - #g Gly Trp Asp Gly Phe         #                15                                                           - Val Glu Phe Phe His Val                                                                 20                                                                - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..23                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - His Asn Leu Leu Pro Trp Met Ile Ser His Gl - #y Gly Gln Glu Glu Phe         #                15                                                           - Leu Ala Phe Ser Leu His Ser                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..23                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #A"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Glu Gly Leu Asp Gly Trp Ile His Gln Gln Gl - #y Gly Trp Ser Thr Leu         #                15                                                           - Ile Glu Asp Asn Ile Pro Gly                                                             20                                                                - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #B"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Glu Glu Leu Phe Arg Asp Gly Val Asn Trp Gl - #y Arg Ile Val Ala             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #B"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Glu Glu Leu Ala Ala Ala Ala Val Asn Trp Gl - #y Arg Ile Val Ala             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #B"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Glu Glu Leu Phe Arg Asp Gly Val Asn Ala Al - #a Ala Ile Val Ala             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #B"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Glu Glu Leu Phe Arg Asp Gly Val Asn Trp Al - #a Arg Ile Val Ala             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #B"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Glu Glu Leu Phe Arg Asp Gly Val Asn Trp Gl - #u Arg Ile Val Ala             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #C"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Trp Ile Gln Asp Asn Gly Gly Trp Asp Ala Ph - #e Val Glu Leu Tyr             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #C"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Trp Ile Leu Ala Ala Gly Gly Trp Asp Ala Ph - #e Val Glu Leu Tyr             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #C"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Trp Ile Gln Asp Ala Gly Gly Trp Asp Ala Ph - #e Val Glu Leu Tyr             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..11                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #C"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Trp Ile Gln Asp Asn Gly Phe Val Glu Leu Ty - #r                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                 #/note= "Peptide fragment fromN:                                                             Figure 14 - #C"                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Trp Ile Gln Asp Asn Gly Gly Trp Asp Ala Ph - #e Val Ala Leu Tyr             #                15                                                           __________________________________________________________________________

What is claimed is:
 1. A purified and isolated full-length Bcl-2associated protein (Bax) or a fragment thereof, wherein said fragmentcomprises a Domain I sequence and/or a Domain II sequence and binds toBcl-2 or Bax, wherein the Domain I sequence consists of amino acids102-112 of SEQ ID NO:2 or amino acids 102-112 of SEQ ID NO:3 and whereinthe Domain II sequence consists of amino acids 151-159 of SEQ ID NO:2 oramino acids 151-159 of SEQ ID NO:3.
 2. A homodimer comprising the Baxprotein or fragment thereof of claim
 1. 3. The Bax protein or fragmentthereof of claim 1, wherein the Bax protein comprises a Bax amino acidsequence as set forth in SEQ ID NO:2, 3, 5, 7, 8 or
 9. 4. The Baxprotein or fragment thereof of claim 1, wherein the Bax proteincomprises a polypeptide having 192 amino acids and a molecular weight of21 kD.
 5. The Bax protein or fragment thereof of claim 4, wherein theprotein consists of the amino acid sequence as shown in SEQ ID NO:2. 6.A purified and isolated mutant full-length Bcl-2 associated protein(Bax) comprising an amino acid substitution or deletion in a Domain Isequence consisting of amino acids 102-112 of SEQ ID NO:2 or amino acids102-112 of SEQ ID NO:3 and/or a Domain II sequence consisting of aminoacids 151-159 of SEQ ID NO:2 or amino acids 151-159 of SEQ ID NO:3,whereby the mutant Bax protein is substantially incapable of binding toBcl-2 or Bax.
 7. A fragment of the mutant Bax protein of claim 6,wherein the fragment comprises the amino acid substitution or deletionin the Domain I sequence and/or the Domain II sequence.
 8. The proteinof claim 6, wherein the glycine present in the Domain I sequence NWGR isreplaced with alanine or glutamic acid.
 9. The fragment of claim 6,wherein the glycine present in the Domain I amino acid sequence NWGR isreplaced with alanine or glutamic acid.
 10. The fragment of claim 1,wherein at least one peptide linkage in the Domain I sequence and/or theDomain II sequence is replaced by a nonpeptide linkage selected from thegroup consisting of: --CH₂ NH--, CH₂ S--, --CH₂ CH₂ --, --CH═CH--,--COCH₂ --, --CH(OH)CH₂ --, and CH₂ SO--.
 11. The fragment of claim 10,wherein the nonpeptide linkage is --CH₂ NH--.
 12. A protein complexwhich comprises a full-length Bax protein or fragment thereof associatedwith Bcl-2, wherein said Bax fragment comprises a Domain I sequenceand/or a Domain II sequence and binds to Bcl-2 or Bax, wherein theDomain I sequence consists of amino acids 102-112 of SEQ ID NO:2 oramino acids 102-112 of SEQ ID NO:3 and wherein the Domain II sequenceconsists of amino acids 151-159 of SEQ ID NO:2 or amino acids 151-159 ofSEQ ID NO:3.
 13. The protein complex of claim 12, wherein the proteincomplex is in the form of a heterodimer.
 14. The protein complex ofclaim 12 wherein the Bax protein comprises a Bax amino acid sequence asset forth in SEQ ID NO:2, 3, 5 or
 7. 15. An isolated polypeptidecomprising a Bax epitope which comprises a sequence of 10 consecutiveamino acids which is identical to a sequence of 10 consecutive aminoacids of SFQ ID NO:2, 3, 5 or 7, wherein the epitome reacts with anantibody specific for a full-length Bax polypeptide.
 16. The isolatedpolypeptide of claim 15 which comprises a sequence of 192 to 218contiguous amino acids which is identical to a naturally-occurringfull-length Bax polypeptide.
 17. The isolated polypeptide of claim 15which comprises a naturally occuring mammalian Bax polypeptide.
 18. Theisolated polypeptide of claim 17 which comprises a human Baxpolypeptide.
 19. The isolated polypeptide of claim 18, which is 192amino acids long and is identical to the full-length Bax polypeptidesequence shown in SEQ ID NO:2.
 20. The isolated polypeptide of claim 18,wherein said polypeptide is 218 amino acids long and consists of aminoacids 1-57 of SEQ ID NO:2 contiguous with SEQ ID NO:5, wherein aminoacid 57 of SEQ ID NO:2 is joined in peptide linkage with amino acid 1 ofSEQ ID NO:5.
 21. A human or murine full-length Bax protein or fragmentthereof encoded by a recombinant polynucleotide in a host cell, whereinsaid fragment comprises a Domain I sequence and/or a Domain II sequenceand binds to Bcl-2 or Bax, wherein the Domain I sequence consists ofamino acids 102-112 of SEQ ID NO:2 or amino acids 102-112 of SEQ ID NO:3and wherein the Domain II sequence consists of amino acids 151-159 ofSEQ ID NO:2 or amino acids 151-159 of SEQ ID NO:3.
 22. The purified andisolated full-length Bax protein or fragment thereof of claim 1 which isisolated from a recombinant host cell comprising a polynucleotideencoding the Bax polypeptide.
 23. A full-length Bax polypeptide or afragment thereof labeled with a radioisotope or fluorescent label,wherein said fragment comprises a Domain I sequence and/or a Domain IIsequence and binds to Bcl-2 or Bax, wherein the Domain I sequenceconsists of amino acids 102-112 of SEQ ID NO:2 or amino acids 102-112 ofSEQ ID NO:3 and wherein the Domain II sequence consists of amino acids151-159 of SEQ ID NO:2 or amino acids 151-159 of SEQ ID NO:3.
 24. Apharmaceutical composition comprising a pharmaceutically effectiveamount of a full-length Bax protein or a fragment thereof, and apharmaceutically suitable carrier, wherein said fragment comprises aDomain I sequence and/or a Domain II sequence and binds to Bcl-2 or Bax,wherein the Domain I sequence consists of amino acids 102-112 of SEQ IDNO:2 or amino acids 102-112 of SEQ ID NO:3 and wherein the Domain IIsequence consists of amino acids 151-159 of SEQ ID NO:2 or amino acids151-159 of SEQ ID NO:3.
 25. A purified and isolated full-length humanBax protein or a fragment thereof, wherein said fragment comprises sevenSer/Thr residues, a Domain I sequence and a Domain II sequence and bindsto Bcl-2 or Bax, wherein the Domain I sequence consists of amino acids102-112 of SEQ ID NO:2 and wherein the Domain II sequence consists ofamino acids 151-159 of SEQ ID NO:2.
 26. A purified and isolatedfull-length murine Bax protein or a fragment thereof, wherein saidfragment comprises seven Ser/Thr residues, a Domain I sequence and aDomain II sequence and binds to Bcl-2 or Bax, wherein the Domain Isequence consists of amino acids 102-112 of SEQ ID NO:3 and wherein theDomain II sequence consists of amino acids 151-159 of SEQ ID NO:3.